Analysis of Endocrine Disrupting Compounds in Food

Analysis of Endocrine Disrupting Compounds in Food

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

Analysis of Endocrine Disrupting Compounds in Food Leo M.L. Nollet EDITOR

A John Wiley & Sons, Inc., Publication

Edition first published 2011 © 2011 Blackwell Publishing Ltd. Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical, and Medical business to form Wiley-Blackwell. Editorial Office 2121 State Avenue, Ames, Iowa 50014-8300, USA For details of our global editorial offices, for customer services, and for information about how to apply for permission to reuse the copyright material in this book, please see our Website at www.wiley.com/ wiley-blackwell. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee code for users of the Transactional Reporting Service is ISBN-13: 978-0-8138-1816-0/2011. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Analysis of endocrine disrupting compounds in food / editor, Leo M.L. Nollet. p. cm. Includes bibliographical references and index. ISBN 978-0-8138-1816-0 (hardback : alk. paper) 1. Food–Analysis. 2. Endocrine disrupting chemicals–Analysis. I. Nollet, Leo M. L., 1948– TX541.A75 2011 664'.07–dc22 2010016639 A catalog record for this book is available from the U.S. Library of Congress. Set in 10 on 12 pt Times by Toppan Best-set Premedia Limited Printed in Singapore Disclaimer The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation warranties of fitness for a particular purpose. No warranty may be created or extended by sales or promotional materials. The advice and strategies contained herein may not be suitable for every situation. This work is sold with the understanding that the publisher is not engaged in rendering legal, accounting, or other professional services. If professional assistance is required, the services of a competent professional person should be sought. Neither the publisher nor the author shall be liable for damages arising herefrom. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. 1

2011

Table of Contents

Preface List of contributors

vii ix

Chapter 1

Endocrine Disrupting Chemicals. What? Where? Guang-Guo Ying

3

Chapter 2

Analysis of PCBs in Food Manuela Melis and Ettore Zuccato

19

Chapter 3

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food Luisa R. Bordajandi, Belén Gómara, and María José González

49

Chapter 4

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities M.J. Gómez, M.A. Martínez-Uroz, M.M. Gómez-Ramos, A. Agüera, and A.R. Fernández-Alba

75

Chapter 5

Pesticides: Herbicides and Fungicides Iván P. Román Falcó, Lorena Vidal, and Antonio Canals

127

Chapter 6

Pesticides: Organophosphates Juan F. García-Reyes, Bienvenida Gilbert-López, and Antonio Molina-Díaz

199

Chapter 7

Phytoestrogens Ashok K. Singh and Leo M.L. Nollet

219

Chapter 8

Mycoestrogens Jean-Denis Bailly

229

Chapter 9

Analysis of Hormones in Food John L. Zhou and Zulin Zhang

243

Chapter 10 Phthalates Jiping Zhu, Rong Wang, Yong-lai Feng, and Xu-liang Cao

255

Chapter 11 Organotin Compounds Analysis Maw-Rong Lee and Chung-Yu Chen

269

Chapter 12 Determination of Heavy Metals in Food by Atomic Spectroscopy Joseph Sneddon

289

v

vi

Contents

Chapter 13 Surfactants Bing Shao

305

Chapter 14 Polybrominated Biphenyls Antonia María Carro-Díaz and Rosa Antonia Lorenzo-Ferreira

325

Chapter 15 Bisphenol A Ana Ballesteros-Gómez and Soledad Rubio

349

Chapter 16 Perfluoroalkylated Substances Leo M.L. Nollet

367

Chapter 17 Flame Retardants D. Lambropoulou, E. Evgenidou, Ch. Christophoridis, E. Bizani, and K. Fytianos

377

Chapter 18 Personal Care Products Guang-Guo Ying

413

Chapter 19 Polycyclic Aromatic Hydrocarbons Peter Šimko

429

Chapter 20 Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene Leo M.L. Nollet

447

Index

471

Preface

Many chemical compounds used in the past, and others still used, may have hormonedisrupting properties. Such chemical compounds may interfere with the normal action of hormones in humans and animals. A great number of these endocrine-disrupting compounds are persistent organic pollutants (POPs). POPs may be found worldwide and in every compartment of the environment: water, air, and soil. Animals and humans may inhale or ingest residues of these chemical compounds. Other sources of endocrine disruptors are accidents and pollution. Analysis of Endocrine Disrupting Compounds in Food provides, first and above all, a unique and comprehensive professional reference source covering most of the recent analytical methodology used to study endocrine-disrupting compounds in food. A broad team of international authors addresses the most recent advances in analysis of endocrine-disrupting chemicals in food. The book further discusses the relationship between chemical compounds and hormone activity. What are the health impacts of different chemical compounds for humans and animals? How are these compounds entering into foodstuffs? While covering conventional (typically lab-based) methods of analysis, the book focuses on leading-edge technologies that have recently been introduced. The book looks at areas such as food quality assurance and safety. The topics of the presence of

persistent organic pollutants; monitoring pesticide and herbicide residues in food; determining heavy and other metals in food; discussing the impact of dioxins, PCBs, PCDFs, and many other suspected chemicals are covered. The book highlights the relevance and importance of sample preparation and cleanup. Applications of gas chromatography, high-pressure liquid chromatography, and related techniques, and the use of biosensors for evaluating the safety and quality of food and agricultural products are discussed. The reader will also find information on the principles and applications of immunodiagnostics and applications in food safety. A unique feature of the book is that the informational tables are structured the same way throughout the book; furthermore, most chapters are also structured similarly. For all their great efforts and their excellent work I thank all of the authors who contributed to this work. It is their efforts that give value to this book. A special thanks is directed to Mark Barrett and Susan Engelken of Wiley-Blackwell for their support. I dedicate this book to my fourth grandchild and first grandson, Naut. I hope he will become a respected and loved man in a green world, a world without endocrine disrupting compounds. Leo M.L. Nollet

Exert your talents, and distinguish yourself, and don’t think of retiring from the world, until the world will be sorry that you retire. (Samuel Johnson) vii

List of Contributors

A. Agüera Pesticide Residue Research Group University of Almería 04120 Almería, Spain Jean-Denis Bailly Mycotoxicology Research Unit National Veterinary School of Toulouse 23 Chemin des Capelles BP 87614 31076 Toulouse Cedex 03, France Ana Ballesteros-Gómez Department of Analytical Chemistry Campus of Rabanales Edificio Anexo Marie Curie University of Córdoba 14071 Córdoba, Spain E. Bizani Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece Luisa R. Bordajandi European Food Safety Authority (EFSA) Unit on Contaminants Largo N. Palli 5/A. 43100 Parma, Italy Antonio Canals Departamento de Química Analítica Nutrición y Bromatología e Instituto Universitario de Materiales Universidad de Alicante P.O. Box 99 03080 Alicante, Spain

Xu-liang Cao Food Research Division Health Canada 251 Sir Frederick Banting Driveway Tunney’s Pasture Ottawa Ontario K1A 0L2 Canada Antonia María Carro-Díaz Dpto. de Química Analítica Nutrición y Bromatología Facultad de Química Instituto de Investigacións e Análises Alimentarios Universidad de Santiago de Compostela Avda. de las Ciencias s/n 15782 Santiago de Compostela, Spain Chung-Yu Chen Department of Chemistry National Chung Hsing University Taichung Taiwan Ch. Christophoridis Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece E. Evgenidou Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece ix

x

Contributors

Iván P. Román Falcó Departamento de Química Analítica Nutrición y Bromatología e Instituto Universitario de Materiales Universidad de Alicante P.O. Box 99 03080 Alicante Spain Yong-lai Feng Exposure and Biomonitoring Division Health Canada 50 Columbine Driveway Tunney’s Pasture Ottawa Ontario K1A 0K9 Canada A.R. Fernández-Alba IMDEA (Instituto Madrileño de Estudios Avanzados) Parque Científico Tecnológico de la Universidad de Alcalá 28805 Alcalá de Henares, Madrid Spain Pesticide Residue Research Group University of Almería 04120 Almería, Spain K. Fytianos Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece

Belén Gómara Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute, CSIC Juan de la Cierva 3, 28006 Madrid Spain

M.J. Gómez IMDEA (Instituto Madrileño de Estudios Avanzados) Parque Científico Tecnológico de la Universidad de Alcalá 28805 Alcalá de Henares, Madrid Spain Department of Chemical Engineering University of Alcala 28771 Alcala de Henares, Madrid Spain

M.M. Gómez-Ramos Pesticide Residue Research Group University of Almería 04120 Almería, Spain

María José González Instrumental Analysis and Environmental Chemistry Department General Organic Chemistry Institute, CSIC Juan de la Cierva 3, 28006 Madrid Spain

Juan F. García-Reyes Analytical Chemistry Research Group Department of Physical and Analytical Chemistry University of Jaen 23071 Jaén, Spain

D. Lambropoulou Department of Chemistry Aristotle University of Thessaloniki 54124 Thessaloniki, Greece

Bienvenida Gilbert-López Analytical Chemistry Research Group Department of Physical and Analytical Chemistry University of Jaen 23071 Jaén, Spain

Maw-Rong Lee Department of Chemistry National Chung Hsing University Taichung Taiwan

Contributors

Rosa Antonia Lorenzo-Ferreira Dpto. de Química Analítica Nutrición y Bromatología Facultad de Química Instituto de Investigacións e Análises Alimentarios Universidad de Santiago de Compostela Avda. de las Ciencias s/n 15782 Santiago de Compostela, Spain M.A. Martínez-Uroz Pesticide Residue Research Group University of Almería 04120 Almería, Spain Manuela Melis Laboratory of Food Toxicology Department of Environmental Health Sciences Mario Negri Institute for Pharmacological Research Via La Masa 19, 20156 Milan, Italy Antonio Molina-Díaz Analytical Chemistry Research Group Department of Physical and Analytical Chemistry University of Jaen 23071 Jaén, Spain

xi

Bing Shao Institute of Nutrition and Food Hygiene Beijing Center for Disease Control and Prevention Beijing 100013, China Peter Šimko Food Research Institute Priemyselna 4 824 75 Bratislava Slovak Republic and Institute of Food Science and Biotechnology Faculty of Chemistry Brno University of Technology Purkynˇ ova 464/118, 612 00 Brno Czech Republic Ashok K. Singh Department of Veterinary Diagnostic Medicine College of Veterinary Medicine University of Minnesota, St Paul Campus 1333 Gortner Ave St Paul, MN 55108 USA Joseph Sneddon Department of Chemistry McNeese State University Lake Charles, LA 70609 USA

Leo M.L. Nollet University College Ghent Member of Ghent University Association Faculty of Applied Engineering Sciences Schoonmeersstraat 52 B9000 Gent Belgium

Lorena Vidal Departamento de Química Analítica Nutrición y Bromatología e Instituto Universitario de Materiales Universidad de Alicante P.O. Box 99 03080 Alicante, Spain

Soledad Rubio Department of Analytical Chemistry Campus of Rabanales Edificio Anexo Marie Curie University of Córdoba 14071 Córdoba, Spain

Rong Wang Exposure and Biomonitoring Division Health Canada 50 Columbine Driveway Tunney’s Pasture Ottawa Ontario K1A 0K9 Canada

xii

Contributors

Guang-Guo Ying State Key Laboratory of Organic Geochemistry Guangzhou Institute of Geochemistry Chinese Academy of Sciences 511 Kehua Street, Tianhe District Guangzhou 510640, China

Jipin Zhu Exposure and Biomonitoring Division Health Canada 50 Columbine Driveway Tunney’s Pasture Ottawa Ontario K1A 0K9 Canada

Zulin Zhang The Macaulay Institute Craigiebuckler Aberdeen, AB15 8QH, UK

Ettore Zuccato Laboratory of Food Toxicology Department of Environmental Health Sciences Mario Negri Institute for Pharmacological Research Via La Masa 19, 20156 Milan, Italy

John L. Zhou School of Life Sciences Science University of Sussex Falmer, Brighton BN1 9QG, UK

Chapter 1 Endocrine Disrupting Chemicals. What? Where? Guang-Guo Ying

Introduction There is a concern that some natural and synthetic chemicals can interfere with the normal functioning of endocrine systems, thus affecting reproduction and development in wildlife and humans. These chemicals are called endocrine disruptors or endocrinedisrupting chemicals (EDCs). Although endocrine disruption has been known since the 1930s (Dodds et al. 1938), this issue has regained attention and generated immense scientific and public interest since 1992 (Colborn and Clement 1992), and especially since the publication of the book Our Stolen Future (Colborn et al. 1996). The chemicals identified or suspected as being endocrine disruptors in the literature include pesticides (e.g., dichlorodiphenyltrichloroethane [DDT], dichlorodiphenyldichloroethylene [DDE], dieldrin, endosulfan), pharmaceuticals (e.g., diethylstilbestrol [DES]) and industrial chemicals or pollutants (e.g., polychlorinated biphenyls [PCBs], dioxins, bisphenol A) (Table 1.1). Since then, many studies have been carried out on endocrine disruption. Some reproductive problems in wildlife and humans have been linked to exposure to these chemicals. Wildlife and humans are exposed daily to these pervasive chemicals that have already caused numerous adverse effects in

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

wildlife and are most likely affecting humans as well. There is compelling evidence regarding the effects of exposure to EDCs on wildlife (Damstra et al. 2002). These include imposex of mollusks by organotin compounds (Alzieu 2000; Gibbs et al. 1990; Horiguchi et al. 1994); developmental abnormalities, demasculinization and feminization of alligators in Florida by organochlorines (Guillette et al. 1994, 2000); and feminization of fish by wastewater effluent from sewage treatment plants and paper mills (Table 1.2) (Jobling et al. 1998; Bortone et al. 1989). There is also evidence that human testicular and breast cancer rates have increased during the last four decades, especially in developed countries (Brown et al. 1986; Hakulinen et al. 1986; Adami et al. 1994; Feuer 1995; Moller 1993; Ries et al. 1991; Wolff et al. 1993). However, except in a few cases (e.g., DES), a causal relation between exposure to chemicals and adverse health effects in humans has not been firmly established. Owing to the scientific evidence and public concern about potential effects on humans and wildlife, the U.S. Congress made amendments to the Safe Drinking Water Act (SDWA) in 1996 and required the U.S. Environmental Protection Agency (U.S. EPA) to develop a screening program for endocrine disruptors (Fenner-Crisp et al. 2000). In April 2000, a meeting of the environment ministers of the G8 group of industrialized countries listed EDCs as one of the high priorities and called for a furtherance of knowledge acquisition 3

4

Analysis of Endocrine Disrupting Compounds in Food

Table 1.1. List of suspected/known EDCs. Classification

Endocrine-Disrupting Chemicals

Pesticides

2,4-D Atrazine Benomyl Carbaryl Chlordane (γ-HCH) Cypermethrin DDT and its metabolites Dicofol Dieldrin/Aldrin Endosulfan Endrin Heptachlor Hexachlorobenzene (HCB) Iprodione

Kepone (chlordecone) Lindane Malathion Mancozeb Methomyl Methoxychlor Mirex Parathion Pentachlorophenol Permethrin Simazine Toxaphene Trifluralin Vinclozolin

Organohalogens

Dioxins and furans PCBs

PBBs and PBDEs 2,4-Dichlorophenol

Alkylphenols

Nonylphenols Octylphenols Pentaphenols

Nonylphenol ethoxylates Octylphenol ethoxylates Butylphenols

Heavy metals

Cadmium Lead

Mercury Arsenic

Organotins

Tributyltin

Triphenyltin (TPhT)

Phthalates

Diethylhexyl phthalate Butyl benzyl phthalate Di-n-butyl phthalate Di-n-pentyl phthalate

Dihexyl phthalate Dipropyl phthalate Dicyclohexyl phthalate Diethyl phthalate

Natural hormones

17β-Estradiol Estrone

Estriol Testosterone

Pharmaceuticals

Ethinyl estradiol Mestranol

Tamoxifen Diethylstilbestrol (DES)

Phytoestrogens

Isoflavonoids Coumestans Lignans

Zearalenone β-sitosterol

Phenols

Bisphenol A

Bisphenol F

Aromatic hydrocarbons

Benzo(a)pyrene Benz(a)anthracene Benzo(b/h)fluoranthene 6-hydroxy-chrysene

Anthracene Pyrene Phenanthrene n-Butyl benzene

on EDCs through jointly planned and implemented projects and international information sharing (Loder 2000). Surveys of some new emerging endocrine disrupting chemicals (e.g., nonylphenol and steroids) in major rivers of some countries have been undertaken (e.g., Naylor et al. 1992; Blackburn et al. 1999; Ahel et al. 2000; Tabata et al. 2001; Kolpin et al. 2002). The U.S. EPA and the Organization of Economic and Cooperative

Development (OECD) have invested considerable resources to develop tiered procedures for the testing and assessment of EDCs (Fenner-Crisp et al. 2000; Huet 2000; Parrott et al. 2001). The U.S. EPA planned to screen 15,000 chemicals for their possible effects as endocrine disruptors in animals and humans (Macilwain 1998). This chapter will give some background information about the endocrine disruption

Endocrine Disrupting Chemicals. What? Where?

5

Table 1.2. Effects associated with exposure to EDCs in wildlife (representative examples). Organism Alligators (Lake Apopka)

Fish (roach, trout) (Rivers in UK) Fish (Rivers in Canada and Finland) Fish (salmon) (Great Lakes) Birds (gulls, bald eagles) (Great Lakes, and California coast)

Chemical(s) DDT and its metabolites (DDD and DDE), dicofol and other pesticides Hormone steroids and nonylphenols from STP effluents Chemicals from pulp and paper mill effluents PCBs, dioxins, and organochlorine pesticides PCBs, DDT, and DDE

Effect

References

Demasculinization, reproductive dysfunction

Guillette et al. (1994) Guillette et al. (1995) Guillette et al. (2000) Pickford et al. (2000)

Feminization, abnormal gonad and gonoduct development Delayed sexual maturity, smaller gonads, reduced fecundity and changed sex steroid levels Abnormal thyroid function

Purdom et al. (1994) Jobling and Sumpter (1993) Jobling et al. (1998) Aaltonen et al. (2000) Karels et al. (1999) McMaster et al. (1995) Munkittrick et al. (1997) Leatherland (1993) Bowerman et al. (2000) Fry et al. (1987) Moccia et al. (1986)

Invertebrates (snails, mussels, clams, oysters)

Tributyltin (TBT)

Feminization, abnormal thyroid function, supernormal clutches, and decreased hatchability Masculinization (imposex)

Sheep

Phytoestrogens

Decreased fertility

issue, EDCs in food, and potential effects associated with exposure to EDCs.

Endocrine-disruption chemicals Endocrine system An endocrine system is found in nearly all animals, including mammals, nonmammalian vertebrates (e.g., fish, amphibians, reptiles and birds) and invertebrates (e.g., snails, lobsters, insects, and other species). Along with the nervous system, the endocrine system is one of the two communication systems that regulate all responses and functions of the body. The endocrine system consists of glands and the hormones they produce that guide the development, growth, reproduction, and behavior of humans and animals. The major endocrine glands of the body include the pituitary, thyroid, parathyroids, adrenals, pancreas, pineal gland, and gonads (ovaries in females and testes in males).

Alzieu (1998, 2000) Gibbs and Bryan (1986) Gibbs et al. (1988) Jha et al. (2000a) Jha et al. (2000b) Morcillo and Porte (2000) Bennetts et al. (1946) Hughes (1988)

Hormones are biochemicals that are produced by endocrine glands in one part of the body, travel through the bloodstream, and cause responses in other parts of the body. They act as chemical messengers and interact with specific receptors in cells to trigger responses and prompt normal biological functions such as growth, reproduction, and development. Hormones generally fall into four main categories: (1) amino acid derivatives, (2) proteins, (3) steroids, and (4) eicosanoids (Lister and van der Kraak 2001). The unifying nature of hormone action is the presence of receptors on target cells, which bind a specific hormone with high affinity and stereospecificity. Steroid and thyroid hormones act by entering target cells and stimulating specific genes. All other hormones bind to receptors on the cell surface and activate second-messenger molecules within the target cells (Raven and Johnson 1999). The body has hundreds of different kinds of receptors; each one is

6

Analysis of Endocrine Disrupting Compounds in Food

Figure 1.1. Functioning of hormone system. VTG, vitellogenin; ER, estrogen receptor; Hsp90, heat shock protein 90 kDa; ERE, estrogen response element.

designed to receive a particular kind of chemical signal. The hormone and its receptor have a “lock-and-key” relationship (Figure 1.1). When a hormone encounters its receptor, they grab hold, engaging in a molecular embrace known as binding. Once joined, the hormone molecule and its receptor trigger the production of particular proteins that turn on the biological activity associated with the hormone. The actions of hormones have two types: organizational and activational (Lister and van der Kraak 2001). The first type of action occurs during critical periods of development and induces permanent effects such as the actions of sex steroids. The second type of action causes only transient changes in a myriad of cellular processes such as the effects of glucagon and insulin on glucose homeostasis. Organizational actions are more important in terms of effect with respect to environmental contaminants (Guillette et al. 1996). Timing of hormone release is often critical for normal function, especially during fetal development (Palanza et al. 1999).

Endocrine disruption The Society of Environmental Toxicology and Chemistry (SETAC) defined endocrine

disruption as follows: “Synthetic, and naturally occurring, chemical substances in the environment are disrupting the normal functions of the endocrine system and its hormones in humans and wildlife” (SETAC 2000). This hypothesis has received much attention in recent years because there is increasing evidence that some chemicals in our environment disrupt the endocrine systems in wildlife as well as humans. There are several ways that chemicals can interfere with the endocrine system (Sonnenschein and Soto 1998). They can mimic or block natural hormones and alter hormonal levels, thus affect the functions that these hormones control. Less direct disruption involves alteration of the body’s ability to produce hormones, interference with the ways hormones travel through the body, and changes in numbers of receptors. Regardless of the situation, having too much or too little of the hormones it needs may cause the endocrine system to function inappropriately. Very subtle disruptions of the endocrine system can result in changes in growth, development, or behavior that can affect the organism itself or the next generation (Guillette et al. 1996; vom Saal et al. 1997; Palanza et al. 1999).

Endocrine Disrupting Chemicals. What? Where?

Hormones play a crucial role in the proper development of the growing fetus. Embryos and fetuses are especially sensitive at particular times to low doses of endocrine disruptors (Guillette et al. 1996; vom Saal et al. 1997; Palanza et al. 1999). Substances that have no effect in an adult can become poisonous in the developing embryo. The timing of exposure may be more important than the dose of the substance. The ultimate effects of endocrine disruption might not be seen until later in life or even until the next generation (Colborn et al. 1996; U.S. EPA 1997).

Endocrine disruptors Endocrine disruptors have received growing attention in public media and the scientific community due to their potential impacts on humans and wildlife. There are several definitions used by scientists and policy makers. In the Organization of Economic and Cooperative Development (OECD), an endocrine-disrupting chemical has been defined as an exogenous substance or mixture that alters the function(s) of the endocrine systems and consequently causes adverse health effects in an intact organism or its progeny or (sub) populations (Lister and Van Der Kraak 2001). An environmental endocrine disruptor was also broadly defined by the U.S. EPA as “an exogenous agent that interferes with the production, release, transport, metabolism, binding, action, or elimination of natural hormones in the body responsible for maintenance of homeostasis and the regulation of developmental processes” (Kavlock et al. 1996). A potential endocrine disruptor can be simply defined as a substance that possesses properties that might be expected to lead to endocrine disruption in an intact organism. An extensive list of the chemicals (Table 1.1) (Colborn et al. 1996; Guillette et al. 1996; Sonnenschein and Soto 1998; U.S. EPA 1997; Depledge and Billninghurst 1999) that have been found to be or are suspected to be capable of disrupting the endocrine

7

systems include many pesticides that are designed to be bioactive (e.g., DDT, vinclozolin, tributyltin [TBT], atrazine), persistent organochlorines (e.g., PCBs, dioxins and furans), alkyl phenols (e.g. nonylphenol and octylphenol), heavy metals (e.g., cadmium, lead, mercury), phytoestrogens (e.g., isoflavoids, lignans, β-sitosterol), and synthetic and natural hormones (e.g., β-estradiol, ethinyl estradiol). Many of these compounds have little in common structurally or in terms of their chemical properties, but they evoke agonist or antagonist responses, possibly through comparable mechanisms of action. These chemicals are released from a wide variety of sources such as intensive agriculture, industrial wastes, mining activity, domestic sewage, and landfills. Suspected EDCs can be found in every division of our environment (air, water, soil, sediment, and biota), in industrial products and household items, and even in the food we eat. They are often found in mixtures, such as effluents from sewage treatment plants, paper mills, and textile factories. It is not clear whether the components in a mixture act additively, synergistically, or antagonistically. EDCs can be classified into the following categories: 1. 2. 3.

4.

5.

6.

Environmental estrogens, for example, methoxychlor, bisphenol A Environmental antiestrogens, for example, dioxin, endosulfan Environmental antiandrogens, for example, vinclozolin, DDE, kraft mill effluent Toxicants that reduce steroid hormone levels, for example, fenarimol and other fungicides, endosulfan Toxicants that affect reproduction primarily through effects on the central nervous system (CNS), for example, dithiocarbamate Toxicants that affect hormone status, for example, cadmium, benzidine-based dyes (Depledge and Billinghurst 1999)

8

Analysis of Endocrine Disrupting Compounds in Food

The chemistry of the potential endocrine disruptors varies greatly, as does potency, that is, the effectiveness in binding and turning on the response. Most endocrine disruptors have very low potency because their chemistry is significantly different from the hormones they mimic. In addition to potency, the potential for a hormonelike effect depends on dose. For most of the endocrine disruptors, the dose–response relationship has not yet been established, especially at the low-dose range, and this may differ from species to species. The risk of endocrine disruptors to humans and wildlife also depends on their behavior and fate in the environment. Chemicals behave differently in different media. For example, nonylphenol had a dissipation halflife of ≤1.2 days in the water column, 28– 104 days in sediment, and 8–13 days on macrophytes in an experimental littoral ecosystem (Liber et al. 1999). Some EDCs (e.g., DDT and PCBs) are ubiquitous and persistent in the environment (Atlas and Giam 1981). They accumulate in the fatty tissue of organisms and increase in concentration as they move up through the food web (biomagnification). Because of their persistence and mobility, they accumulate in organisms and harm

species far from their original source. In order to assess the risks, it is necessary to carry out monitoring of those chemicals possessing endocrine-disrupting characteristics in environmental media and foods we eat.

Human exposure of endocrinedisrupting chemicals Endocrine-disrupting chemicals need to enter an organism before they can disrupt its endocrine system. Humans can be exposed in a variety of ways (Figure 1.2): the food we eat, the air we breathe and the particles or vapors it contains, the pharmaceuticals we ingest for medical reasons, the water we drink, the soil we accidentally or intentionally eat, and in utero exposure from the mother ’s body burden (Crisp et al. 1998). For fat-soluble chemicals such as PCBs, for example, food is the major source for people. Dairy products, meat, and processed foods are all major contributors. Breast milk is also a contributor (Table 1.3). These fat-soluble chemicals remain in the body for a long time, and their accumulation early in life contributes significantly (approximately 15%) to the adult body burden (Patandin et al. 1999).

Dermal exposure Cosmetics Body creams Deodorants

Inhalation exposure PAHs, PBDEs Plasticizers Heavy metals

Accumulation of lipophilic chemicals (DDT/DDE, PCB, PBDE)

Oral exposure Food contaminants Plasticizers PAHs, organochlorines Pesticides or Fungicides Heavy metals

Transfer from mother to fetus or to amniotic fluid or both

Figure 1.2. Routes of human exposure to chemicals.

Transfer of lipophilic chemicals to offspring by breast feeding

9

1973 1995–1997 1972

New South Wales, Australia

Germany

Japan

2002 2000 1996 1997–1998

China

India

Canada

UK

0.006–0.27 (0.071)

α-HCH

1.6–10.67 (5.43) 0.042–0.969 (0.21)

0.004–0.25 (0.04)

0.001–4.4 (0.345)

β-HCH

(0.043)

(0.043)

(0.001)

(0.081)

0.0039–0.0228 (0.0136)

0.003–0.53 (0.08)

0.06–2.37 (0.468)

0.06–0.51 (0.24)

0.93–8.26 (2.22)

0.08–13.2 (2.04) 0.016–7.6 (0.411)

HCBa

ΣDDT

(0.47)

(0.47)

(0.43)

(2.1)

0.77–4.01 (2.224) 0.0813–1.119 (0.28)

0.027–1.54 (0.24)

0.45–5.31 (1.92)

0.48–3.24 (1.38)

3.26–21 (8.6)

0.6–23.2 (5.64) 0.156–4.86 (1.185)

(0.25)

(0.030)

(0.042)

0.076-0.385 (0.2)

0.118-1.81 (0.55)

0.039–1.571 (0.5)

PCBs

References

Tanabe and Kunisue, 2007

Tanabe and Kunisue, 2007

Tanabe and Kunisue, 2007

Tanabe and Kunisue, 2007

Konishi et al. (2001)

Konishi et al. (2001)

Schade and Heinzow (1998)

Siyali (1973)

Stacey et al. (1985)

Miller and Fox (1973)

Quinsey et al. (1995)

Newton and Greene (1972)

Concentration range and mean in parentheses. HCH, hexachlorocyclohexane isomers (α, β-HCH); HCB, hexachlorobenze; ΣDDT, dichlorodiphenyltrichloroethane (DDT and its metabolite DDE); PCBs, polychlorinated biphenyls.

a

1979–1980

Western Australia

1998

1971–1972

1990

1970

Year

Queensland, Australia

Victoria, Australia

Location

Table 1.3. Comparison of levels of selected organochlorines (μg/g fat) in breast milk of women from Australia and other countries.

10

Analysis of Endocrine Disrupting Compounds in Food

Table 1.4. Concentrations of some EDCs in foods. Compounds

Concentrationsa

Food

References

0.1–19.4 μg/kg

Guenther et al. (2002)

13.4–56.3 (32) ng/ml 5.8–235.8 ng/g wet weight

Ademollo et al. (2008) Lu et al. (2007)

5–1220 (147) ng/g wet weight

Ferrara et al. (2008)

Canned fish

2–59 ng/g

Various food (fruit and vegetables, meat)

0.5–384 ng/g

Podlipna and Cichna-Markl (2007) Ballesteros-Gomez et al. (2009)

DEHP

Wine


Del Carlo et al. (2008)

PCBs

Fish from River Nestos, Greece

3.60–27.85 ng/g wet weight

Christoforidis et al. (2008)

4-MBC

River fish in Switzerland Lake fish in Switzerland

50–1800 (420) ng/g lipid weight

Buser et al. (2006)

OC

River fish in Switzerland Lake fish in Switzerland

4-NP

BPA

60 different foodstuffs in Germany Human milk in Italy 25 different types of food in Taiwan Seafood from Tyrrhenian Sea

<20–170 (86) ng/g lipid weight 40–2400 (630) ng/g lipid weight

Buser et al. (2006)

nd

a

Minimum to maximum (mean). 4-NP, 4-nonylphenols; BPA, bisphenol A; DEHP, di-2-ethylhexyl phthalate; PCBs, polychlorinated biphenyls; 4-MBC, 4-methylbenzylidene; OC, octyocrylene; nd, no data.

Various endocrine-disrupting chemicals have been detected in foods, including persistent organic pollutants such as polychlorinated biphenyls and organochlorine pesticides, and emerging contaminants such as 4-nonylphenols, sunscreen agents, and bisphenol A (Tables 1.3 and 1.4). Some contaminants such as 4-nonylphenols have been found to be ubiquitous in various foodstuffs (Guenther et al. 2002). These chemicals may accumulate in the human body and cause endocrine-disrupting effects.

Effects associated with exposure to endocrine-disrupting chemicals Wildlife A variety of reproductive and developmental effects in wildlife have been attributed to exposure to EDCs (see Table 1.2). This includes changes of sex, population decline, increase in cancers, reduced reproductive

function and disorders, disrupted immune and nervous systems, as well as abnormal behavior (U.S. EPA 1997; Jimenez 1997; Depledge and Billinghurst 1999; Damstra et al. 2002). These adverse effects have been reported in various species including invertebrates, fish, reptiles, birds, and mammals. Marine invertebrates The best-documented cases of endocrine disruption in invertebrates are in mollusks exposed to organotin compounds (e.g., tributyltin [TBT]) contained in antifouling paints. Since 1971, many studies have reported detrimental effects of TBT on biota including high larval mortality and severe malformations of shells in oysters (Alzieu et al. 1986; Alzieu 1991); imposex in gastropods (Gibbs et al. 1990; Horiguchi et al. 1994) and dogwhelks (Bryan et al. 1986); growth retardation in mussels (Salazar and Salazar 1991)

Endocrine Disrupting Chemicals. What? Where?

and microalgae (Beaumont and Newman 1986); and deformities in fiddler crabs (Weis et al. 1987). These effects have been observed at exposure concentrations as low as 1 ng/L (Gibbs et al. 1988; Alzieu 2000). The imposex phenomenon is currently the only example of chemical-mediated endocrine disruption that has resulted in an effect at the population level. There have also been reports on the endocrine disruption in crustaceans by trace levels of metals (Cd, Zn) and polychlorinated biphenyls (PCBs) (Bodar et al. 1990; Kristoforova et al. 1984). Fish Endocrine disruption in fish has been widely studied, and the observed reproductive effects include feminization, vitellogenisis, impaired reproductive performance, altered thyroid function, decreased fertility, and decreases in population through exposure to pesticides, PCBs, polyaromatic hydrocarbons (PAHs), nonylphenols, hormone steroids, and phytosterols (U.S. EPA 1997). Widespread sexual disruption in wild fish in the United Kingdom has been found due to exposure to the discharges from sewage treatment plants that contain estrogenic chemicals such as nonylphenol and hormone steroids (Jobling et al. 1998; Sumpter 1998; Tyler and Routledge 1998). In fish, the threshold concentrations for an estrogenic response to a 3-week exposure to 17α-ethynylestradiol, 17β-estradiol, nonylphenol, and octylphenol were 0.1 ng/L, 1 ng/L, 10 μg/L, and 3 μg/L, respectively (Purdom et al. 1994; Routledge et al. 1998; Jobling et al. 1995). The levels of these EDCs in some English rivers are well above the threshold concentrations (Tyler and Routledge 1998). A number of abnormalities, including reductions in gonadal size, delayed sexual maturation, and reduced expression of secondary sexual characteristics, have been observed in white sucker exposed to bleached kraft mill effluent in Canada (McMaster et al. 1991, 1992;

11

Munkittrick et al. 1991). Organochlorines have been implicated in a number of developmental and reproductive abnormalities and altered thyroid function in fish in the Great Lakes of North America (U.S. EPA 1997; Leatherland 1992). Reptiles Alligators in Lake Apopka in Florida were extensively studied and are the subject of a well-documented case of endocrine disruption. The population of alligators declined in the 1980s following a major spill of dicofol. The lake has also received other pesticides from nearby agricultural land (Guillette et al. 2000). Abnormalities ranging from low hatchability of eggs to morphological and physiological deficits in both males and females have been reported (Guillette et al. 1994). These were attributed to endocrine disruption caused by embryonic exposure to elevated tissue concentrations of DDT and its metabolites or other pesticides, as well as an embryotoxic effect of high contaminant concentrations in the eggs (Guillette et al. 2000). Birds The phenomena of eggshell thinning, altered thyroid function, and supernormal clutches (female–female pairing) in wild birds have been attributed to endocrine disruption (U.S. EPA 1997; Fry and Toone 1981). Thin eggshells of birds have been widely reported in the past and were caused by DDE (Dawson 2000). Fry and Toone (1981) suggested that DDT-induced feminization of male gull embryos may be responsible for the biased sex ratio. This resulted in greater numbers of supernormal clutches in western gulls on Santa Barbara Island, California. Low hatching success rates and abnormal thyroid in fish-eating birds (gulls, bald eagles) in the Great Lakes were also linked to organochlorines (DDT, PCBs) (U.S. EPA 1997).

12

Analysis of Endocrine Disrupting Compounds in Food

Mammals Reproductive problems have been found in male Florida panthers, grey seals and common seals and attributed to pollution by PCBs and other organochlorine chemicals (Reijinders 1986; Facemire et al. 1995; U.S. EPA 1997). Poor reproduction of common seals in the western part of the Wadden Sea, the Netherlands, was reported and believed to be caused by pollutants (e.g., PCBs) carried from the Rhine River (Reijinder 1986).

Humans Endocrine disruptors have been linked to a range of human health problems, including increased incidence of testicular, prostate, and female breast cancer; decreased semen quality and counts; increased frequency of cryptorchidism and hypospadias; increased incidence of polycystic ovaries in women; endometriosis; altered physical and mental development (IEH 1995; Colborn et al. 1996; U.S. EPA 1997; Foster 2001). With few exceptions (e.g., DES), a direct causal relationship between exposure to a specific chemical and an adverse effect on human health via an endocrine disruption mechanism has not been established, although some laboratory studies on animals showed that some chemicals might be responsible for the reported reproductive, developmental, and carcinogenic effects in humans. The best-known example of endocrine disruption in humans is the drug DES, a synthetic estrogen that was prescribed to many women in an effort to prevent miscarriage between the late 1940s and early 1970s. Not only did DES not prevent miscarriage, it also had many harmful effects on the children of many of these women. The effects of DES in the offspring of these women were not only cancers, but also birth defects of the uterus and ovaries, reproductive organ dysfunction, and immune suppression (IEH 1995; Colborn et al. 1996; U.S. EPA 1997).

Observations in humans from exposure to contaminated cooking oil in two incidents in Asia provide another good example of how EDCs affect human health (Rogan 1982; Yu et al. 1994; Guo et al. 2000; Aoki et al. 2001). In 1968 in Northern Kyushu in Japan, about 2000 people were poisoned by PCBs and polychlorinated dibenzofurans (PCDFs) that contaminated rice oil. Their condition was named Yusho disease. A similar poisoning by PCBs in Taiwan in 1979 was named YuCheng disease. The symptoms of these two diseases in exposed people were dermal and ocular lesions, irregular menstrual cycles, and altered immune responses. Further studies showed that children born to mothers after the event had impaired cognitive development, intrauterine growth retardation, and dysmorphic and hyperpigmented skin and nails. Prenatally exposed boys had sperm with abnormal morphology, reduced motility, and reduced strength. Recent studies have shown the possible impacts of endocrine disruptors on intelligence and behavior of humans (Laessig et al. 1999; Porter et al. 1999). During the 9 months between conception and birth, the fetal brain is developing and guided by natural chemical signals, including hormone systems; therefore, it is vulnerable to endocrine disruption. Jacobson and Jacobson (1996) found significant learning and attention problems in children of women who had eaten contaminated fish from Lake Michigan in the 6 years prior to pregnancy. The fish from Lake Michigan contained significant levels of PCBs and other contaminants, which had effects on their children. In a similar study by Lonkey et al. (1996), measurable neurobehavioral deficits in the newborn children of women who had eaten the equivalent of 40 pounds of Lake Ontario salmon in a lifetime were observed. The cases discussed above have shown that adverse effects on human health are possible through exposure to some endocrine disruptors. The released “Global Assessment

Endocrine Disrupting Chemicals. What? Where?

of the State of the Science of Endocrine Disruptors” by Damstra et al. (2002) concluded that “studies examining EDC-induced effects in humans have yielded inconsistent and inconclusive results, which is responsible for the overall data being classified as ‘weak’. This classification is not meant to downplay the potential effects of EDCs; rather, it highlights the need for more rigorous studies.” The only evidence showing that humans are susceptible to EDCs is currently provided by studies of high exposure levels. More evidence has recently emerged that exposure to EDCs such as phthalates, PCBs, and mercury can disrupt reproductive and developmental processes (Barrett 2005; Hood 2005). However, many uncertainties about endocrine disruptors are of concern. For example, how great is the effect of EDCs on human health, especially at low exposure levels? What are the unobserved effects of the EDCs? The presently available evidence warrants further research.

Summary Many chemicals can potentially disrupt normal function of reproductive systems in wildlife and humans. These endocrine disruptors could enter food chains through various pathways. Based on the precautionary principle, it is necessary to monitor these chemicals in order to protect human health. Due to the complex nature of food matrices, it is crucial to have sensitive and selective analytical methods for the determination of EDCs in food. We will discuss in detail the extraction and instrumental analysis in the following chapters.

References Aaltonen, T.M., Jokinen, E.I., Lappivaara, J., Markkula, S.E., Salo, H.M., Leppanen, H., and Lammi, R. (2000). Effects of primary- and secondary-treated bleached kraft mill effluents on the immune system and physiological parameters of roach. Aquatic Toxicology 51, 55–67.

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Adami, H.O., Bergstron, R., Mohner, M., Zatonski, W., Storm, H., Ekbom, A., Tretli, S., Teppo, L., Ziegler, H., Rahu, M., Gurevicius, R., and Stengrevics, A. (1994). Testicular cancer in nine northern European countries. International Journal of Cancer 59, 33–38. Ademollo, N., Ferrara, F., Delise, M., Fabietti, F., and Funari, E. (2008). Nonylphenol and octylphenol in human breast milk. Environment International 34, 984–987. Ahel, M., Molnar, E., Ibric, S., and Giger, W. (2000). Estrogenic metabolites of alkylohenol olyethoxylates in secondary sewage effluents and rivers. Water Science and Technology 42(7–8), 15–22. Alzieu, C. (1991). Environmental problems caused by TBT in France: Assessment, regulations, and prospects. Marine Environment Research 32, 7–18. Alzieu, C. (1998). Tributyltin: Case study of a chronic contaminant in the coastal environment. Ocean & Coastal Management 40, 23–36. Alzieu, C. (2000). Impact of tributyltin on marine invertebrates. Ecotoxicology 9, 71–76. Alzieu, C., Sanjuan, J., Deltreil, J.P., and Bovel, M. (1986). Tin contamination in Arcachon Bay: Effects on oyster shell abnormalities. Marine Pollution Bulletin 20, 22–26. Aoki, Y. (2001). Polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans as endocrine disrupters–what we have learned from Yusho disease. Environmental Research 86(1), 2–11. Atlas, E. and Giam, C.S. (1981). Global transport of organic pollutants: Ambient concentrations in the remote marine atmosphere. Science 211, 163–165. Ballesteros-Gomez, A., Rubio, S., Perez-Bendito, D. (2009). Analytical methods for the determination of bisphenol A in food. Journal of Chromatography A 1216, 449–469. Barrett, J. (2005). Phthalates and baby boys: Potential disruption of human genital development. Environmental Health Perspectives 113, A542A544. Beaumont, A.R. and Newman, P.B. (1986). Low levels of tributyltin reduce growth of marine micro-algae. Marine Pollution Bulletin 17, 257–461. Bennetts, H., Underwood, E., and Shier, F. (1946). A specific breeding problem of sheep on subterranean clover pastures in Western Australia. Australian Veterinary Journal 22, 2–12. Blackburn, M.A., Kirby, S.J., and Waldock, M.J. (1999). Concentrations of alkylphenol polyethoxylates entering UK estuaries. Marine Pollution Bulletin 38(2), 109–118. Bodar, C.W.M., Voogt, P.A., and Zandee, D.I. (1990). Ecdysteroids in Daphnia magna: Their role in moulting and reproduction and their levels upon exposure to cadmium. Aquatic Toxicology 17, 339–350. Bortone, S.A., Davis, W.P., and Bundrick, C.M. (1989). Morphological and behavioural characters in mosquito fish as potential bioindicators of exposure to kraft mill effluent. Bulletin of Environmental Contamination Toxicology 43, 370–377.

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Analysis of Endocrine Disrupting Compounds in Food

Bowerman, W.W., Best, D.A., Grubb, T.G., Sikarskie, G., and Giesy, J.P. (2000). Assessment of environmental endocrine disruptors in bald eagles of the Great Lakes. Chemospere 41, 1569–1574. Brown, L.M., Pottern, L.M., Hoover, R.N., Devesa, S.S., Aselton, P., and Flannery, T. (1986). Testicular cancer in the U.S. trends in incidence and mortality. International Journal of Epidemiology 15, 164–170. Bryan, G.W., Gibbs, P.E., Hummerstone, L.G., and Burt, G.R. (1986). The decline of the gastropod Nucella lapillus around southwest England: Evidence for the effect of tributyltin from antifouling paints. Journal of Marine Biological Association of UK 66, 611–640. Buser, H.R., Balmer, M.E., Schmid, P., and Kohler, M. (2006). Occurrence of UV filters 4-methylbenzylidene camphor and octocrylene in fish from various Swiss rivers with inputs from wastewater treatment plants. Environmental Science and Technology 40, 1427–1431. Christoforidis, A., Stamatis, N., Schmieder, K., and Tsachalidis, E. (2008). Organochlorine and mercury contamination in fish tissues from the River Nestos, Greece. Chemosphere 70, 694–702. Colborn, T. and Clement, C. (1992). Chemically induced alterations in sexual development: The wildlife / human connection. In: Colburn, T. and Clement, C., eds., Advances in Modern Environmental Toxicology, vol. 21. Princeton Scientific; Princeton, NJ. Colborn, T., Dumanoski, D., and Myers, J.P. (1996). Our Stolen Future. Plume/Penguin Books; New York. Crisp, T.M., Clegg, E.D., Cooper, R.L., Wood, W.P., Anderson, D.G., Baetcke, K.P., Hoffmann, J.L., Morrow, M.S., Rodier, D.J., Schaeffer, J.E., Touart, L.W., Zeeman, M.G., and Patel, Y.M. (1998). Environmental endocrine disruption: An effects assessment and analysis. Environmental Health Perspectives, 106(suppl 1), 11–56. Damstra, T., Barlow, S., Bergman, A., Kavlock, R., and Van Der Kraak, G. (2002). Global Assessment of the State of the Science of Endocrine Disruptors. International Programme on Chemical Safety, World Health Organisation. Available at http://www.who.int/ pcs. Dawson, A. (2000). Mechanisms of endocrine disruption with particular reference to occurrence in avian wildlife: A review. Ecotoxicology 9, 59–69. Del Carlo, M., Pepe, A., Sacchetti, G., Compagnone, D., Mastrocola, D., and Cichelli, A. (2008). Determination of phthalate esters in wine using solid-phase extraction and gas chromatography-mass spectrometry. Food Chemistry 111, 771–777. Depledge, M. and Billinghurst, Z. (1999). Ecological significance of endocrine disruption in marine invertebrates. Marine Pollution Bulletin 39(1–12), 32– 38. Dodds, E.C., Goldberg, L., Lawson, W., and Robinson, R. (1938). Oestrogenic activity of certain synthetic compounds. Nature 141, 247–248. Facemire, C.F., Gross, T.S., and Guillette, L.J. (1995). Reproductive impairment in the Florida panther: Nature or nurture? Environmental Health Perspectives 103, 79–86.

Fenner-Crisp, P.A., Maciorowski, A.F., and Timm, G.E. (2000). The endocrine disruptor screening program developed by the U.S. Environmental Protection Agency. Ecotoxicolgy 9, 85–91. Ferrara, F., Ademollo, N., Delise, M., Fabietti, F., and Funari, E. (2008). Alkylphenols and their ethoxylates in seafood from the Tyrrhenian Sea. Chemosphere 72, 1279–1285. Feuer, E.J. (1995). Incidence of testicular cancer in US men (Stat Bite). Journal of National Cancer Institute 87, 405. Foster, W.G. (2001). Endocrine disruption and human reproductive effects: An overview. Water Quality Research Journal of Canada 36(2), 253–271. Fry, D.M. and Toone, C.K. (1981). DDT-induced feminisation of gull embryos. Science 213, 922–924. Fry, D.M., Toone, C.K., Speich, S.M., and Peard, R.J. (1987). Sex ratio skew and breeding patterns of gulls: Demographic and toxicological considerations. Studies in Avian Biology. 10, 26–43. Gibbs, P.E. and Bryan, G.W. (1986). Reproductive failure in populations of the dog-whelk, Nucella lapillus, caused by imposex induced by tributyltin from antifouling paints. Journal of Marine Biological Association of UK 66, 767–777. Gibbs, P.E., Bryan, G.W., Pascoe, P.L., and Burt, G.R. (1990). Reproductive abnormalities in female Ocenebra erinacea (Gastropoda) resulting from tributyltin-induced imposex. Journal of Marine Biological Association of United Kingdom 70, 639–656. Gibbs, P.E., Pascoe, P.L., and Burt, G.R. (1988). Sex change in the female dog-whelk Nucella lapillus, induced by tributyltin from antifouling paints. Journal of the Marine Biological Association of the United Kingdom 68, 715–731. Guenther, K., Heinke, V., Thiele, B., Kleist, E., Prast, H., and Raecker, T. (2002). Endocrine disrupting nonylphenols are ubiquitous in food. Environmental Science and Technology 36, 1676–1680. Guillette, L.J., Arnold, S.F., and McLachlan, J.A. (1996). Ecoestrogens and embryos—is there a scientific basis for concern? Animal Reproduction Science 42, 13–24. Guillette, L.J., Crain, D.A., Gunderson, M.P., Kools, S.A., Milnes, M.R., Orlando, E.F., Rooney, A.A., and Woodward, A.R. (2000). Alligators and endocrine disrupting contaminants: A current perspective. American Zoology 40, 438–452. Guillette, L.J., Gross, T.S., Gross, D.A., Rooney, A.A., and Percival, H.F. (1995). Gonadal steroidogenesis in vitro from juvenile alligators obtained from contaminated or control lakes. Environmental Health Perspectives 103(suppl 4), 31–36. Guillette, L.J., Gross, T.S., Masson, G.R., Matter, J.M., Percival, H.F., and Woodward, A.R. (1994). Developmental abnormalities of gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environmental Health Perspectives 102, 680–688. Guo, Y., Hsu, P.C., Hsu, C.C., and Lambert, G.H. (2000). Semen quality after prenatal exosure to polychlori-

Endocrine Disrupting Chemicals. What? Where?

nated biphenyls and dibenzofurans. The Lancet 356, 1240–1241. Hakulinen, T., Andersen, A.A., Malker, B., Rikkala, E., Shou, G., and Tulinius, H. (1986). Trends in cancer incidence in the Nordic countries. A collaborative study of the five Nordic Cancer Registries. Acta Pathologica Microbiologica Immunologica Scandinavia 94(Section A suppl) 288, 1–151. Hood, E. (2005). Growth spurt for EDC recognition. Environmental Health Perspectives 113, A522– A544. Horiguchi, T., Shiraishi, H., Shimizu, M., and Morita, M. (1994). Imposex and organotin compounds in Thais claviger and T. bronni in Japan. Journal of Marine Biological Association of UK 74, 651–669. Huet, M. (2000). OECD activity on endocrine disrupters test guidelines development. Ecotoxicology 9, 77– 84. Hughes, C.L. (1988). Phytochemical mimicry of reproductive hormones and modulation of herbivore fertility by phytoestrogens. Environmental Health Perspectives 78, 171–175. Institute for Environment and Health (IEH). (1995). Environmental oestrogens: Consequences to human health and wildlife. Assessment A1. Institute for Environment and Health; Leicester, United Kingdom. Jacobson, J.L. and Jacobson, S.W. (1996). Intellectual impairment in children exposed to polychlorinated biphenyls in utero. The New England Journal of Medicine 335, 783–789. Jha, A.N., Hagger, J.A., and Hill, S.J. (2000a). Tributyltin induces cytogentic damage in the early life stages of the marine mussel, Mytilus edulis. Environmental and Molecular Mutagenesis 35, 343–350. Jha, A.N., Hagger, J.A., Hill, S.J., and Depledge, M.H. (2000b). Genotoxic, cytotoxic and developmental effects of tributyltin oxide (TBTO): An integrated approach to the evaluation of the relative sensitivities of two marine species. Marine Environmental Research 50, 565–573. Jimenez, B. (1997). Environmental effects of endocrine disruptors and current methodologies for assessing wildlife health effects. Trends in Analytical Chemistry 16(10), 596–606. Jobling, S., Nolan, M., Tyler, C.R., Brighty, G., and Sumpter, J.P. (1998). Widespread sexual disruption in wild fish. Environmental Science and Technology 32, 2498–2506. Jobling, S., Reynolds, T., White, R., Parker, M. G., and Sumpter, J. P. (1995). A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environmental Health Perspectives 103, 582–587. Jobling, S. and Sumpter, J.P. (1993). Detergent components in sewage effluents are weakly oestrogenic to fish: an in vitro study using rainbow trout (Oncorhynchus mykiss) hepatocytes. Aquatic Toxicology 27, 361–372. Karels, A., Soimasuo, M., and Oikari, A. (1999). Effects of pulp and paper mill effluents on reproduction, bile conjugates and liver MFO (mixed function oxygenase)

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activity in fish at southern lake Saimaa, Finland. Water Science and Techology 40(11–12), 109–114. Kavlock, J.R., Daston, G.P., DeRosa, C., Fenner-Crisp, P., Gray, L.E., Kaattari, S., Lucier, G., Luster, M., Mac, M.J., Maczka, C., Miller, R., Moore, J., Rolland, R., Scott, G., Sheehan, M., Sink, T., and Tilson, H.A. (1996). Research needs for risk assessment of health and environmental effects of endocrine disruptors: A report of the US sponsored workshop. Environmental Health Perspectives 104(suppl 4), 715–740. Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., and Buxton, H.T. (2002). Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: A national reconnaissance. Environmental Science and Technology 36, 1202–1211. Konishi, Y., Kuwabara, K., and Hori, S. (2001). Continuous surveillance of organochlorine compounds in human breast milk from 1972 to 1998 in Osaka, Japan. Archives of Environmental Contamination Toxicology 40, 571–578. Kristoforova, N.K., Gnezdilova, S.M., and Vlasova, G.A. (1984). Effects of cadmium on gametogenesis and offspring of the sea urchin Strongylocentrotus intermedius. Marine Ecology and Progressive Series 17, 9–14. Laessig, S.A., McCarthy, M.M., and Silbergeld, E.K. (1999). Neurotoxic effects of endocrine disruptors. Current Opinions in Neurology 12, 745–751. Leatherland, J. (1992). Endocrine and reproductive function in Great lakes salmon. In: Colborn, T. and Clement, C, eds., Chemically Induced Alterations in Sexual and Functional Development: The Wildlife/ Human Connection, pp. 12–145. Princeton Scientific Publishing; Princeton, NJ. Leatherland, J.F. (1993). Field observations on reproductive and developmental dysfunction in introduced and native salmonids from the Great Lakes. Journal of Great Lakes Research 19(4), 737–751. Liber, K., Knuth, M.L., and Stay, F.S. (1999). An integrated evaluation of the persistence and effects of 4-nonylphenol in an experimental littoral ecosystem. Environmental Toxicology and Chemistry 18, 357–362. Lister, A.L. and Van Der Kraak, G.J. (2001). Endocrine disruption: Why is it so complicated? Water Quality Research Journal of Canada 36(2), 175–190. Loder, N. (2000). Royal Society warns on hormone disrupters. Nature 406, 4. Lonkey, E., Reihman, J., Darvill, T., Mather, J., and Daly, H. (1996). Neonatal behaviour assessment scale performance in humans influenced by maternal consumption of environmentally contaminated Lake Ontario fish. Journal of Great Lakes Research 22, 198–212. Lu, Y.Y., Chen, M.L., Sung, F.C., Wang, P.S.G., and Mao, I.F. (2007). Daily intake of 4-nonylphenol in Taiwanese. Environment International 33, 903– 910. Macilwain, C. (1998). US panel split on endocrine disruptors. Nature 395, 828. McMaster, M.E., Portt, C.B., Munkittrick, K.R., and

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Analysis of Endocrine Disrupting Compounds in Food

Dixon, D.G. (1992). Milt characteristics, reproductive performance and larval survival and development of white sucker exposed to bleached kraft mill effluent. Ecotoxicolgy and Environmental Safety 23, 103–117. McMaster, M.E., Van Der Kraak, G.J., and Munkittrick, K.R. (1995). Exposure to bleached kraft pulp mill effluent reduces the steroid biosynthetic capacity of white sucker ovarian follicles. Comparative Biochemistry and Physiology 112C(2), 169–178. McMaster, M.E., Van Der Kraak, G.J., Prott, C.B., Munkittrick, K.R., Silbley, P.K., Smith, I.R., and Dixon, D.G. (1991). Changes in hepatic mixed function oxygenase (MFO) activity, plasma steroid levels and age at maturity of a white sucker (Catostomus commersoni) population exposed to bleached kraft pulp mill effluent. Aquatic Toxicology 21, 199–218. Miller, G.J. and Fox, J.A. (1973). Chlorinated hydrocarbon pesticide residues in Queensland human milks. Medical Journal of Australia 2, 261–264. Moccia, R.D., Fox, G.A., and Britton, A. (1986). A quantitative assessment of thyroid histopathology of herring gulls (Larus argentatus) from the Great Lakes and a hypothesis on the causal role of environmental contaminants. Journal of Wildlife Disease 22, 66–70. Moller, H. (1993). Clues to the aetiology of testicular germ cell tumours from descriptive epidemiology. European Urology 23, 8–15. Morcillo, Y. and Porte, C. (2000). Evidence of endocrine disruption in clams–Ruditapes decussate–transplanted to a tributyltin-polluted environment. Environmental Pollution 107, 47–52. Munkittrick, K.R., Portt, C.B., Van Der Kraak, G.J., Smith, I.R., and Rokosh, D.A. (1991). Impact of bleach kraft mill effluent on population characteristics, liver MFO activity and serum steroid levels of a Lake Superior white sucker (Catostomus commersoni) population. Canadian Journal of Fish and Aquatic Science 48, 232–243. Munkittrick, K.R., Servos, M.R., Carey, J.H., and Van Der Kraak, G.J. (1997). Environmental impacts of pulp and paper wastewater: evidence for a reduction in environmental effects at North American pulp mills since 1992. Water Science and Techology 35(2–3), 329–338. Naylor, C.G., Mieure, J.P., Adams, W.J., Weeks, J.A., Castaldi, F.J., Ogle, L.D., and Romano, R.R. (1992). Alkylphenol ethoxylates in the environment. Journal of the American Oil Chemists’ Society 69(7), 695–703. Newton, K.G. and Greene, N.C. (1972). Organochlorine pesticide residue levels in human milk–Victoria, Australia–1970. Pesticide Monitoring Journal 6(1), 4–8. Palanza, P., Morellini, F., Parmigiani, S., and vom Saal, F. S. (1999). Prenatal exposure to endocrine disrupting chemicals: Effects on behavioural development. Neuroscience and Biobehavioral Reviews 23, 1011–1027. Parrott, J., Wade, M., Timm, G., and Brown, S. (2001). An overview of testing procedures and approaches for identifying endocrine disrupting substances. Water Quality Research Journal of Canada 36(2), 273–291.

Patandin, S., Dagnelie, P.C., Mulder, P.G.H., Op de Coul, E., van der Veen, J.E., Weisglas-Kuperus, N., and Sauer, P.J.J. (1999). Dietary exposure to polychlorinated biphenyls and dioxins from infancy until adulthood: A comparison between breast-feeding, toddler and long-term exposure. Environmental Health Perspectives, 107, 45–51. Pickford, D.B., Guillette, L. J., Crain, D.A., Rooney, A.A., and Woodward, A.R. (2000). Plasma dihydrotestosterone concentrations and phallus size in juvenile American alligators (A. mississippiensis) from contaminated and reference populations. Journal of Herpetology 34(2), 233–239. Podlipna, D., and Cichna-Markl, M. (2007). Determination of bisphenol A in canned fish by sol-gel immunoaffinity chromatography, HPLC and fluorescence detection. European Food Research Technology 224, 629–634. Porter, W.P., Jaeger, J.W., and Carlson, I.H. (1999). Endocrine, immune and behavioural effects of aldicarb (carbamate), atrazine (triazine) and nitrate (fertilizer) mixtures at groundwater concentrations. Toxicology and Industrial Health 15, 133– 150. Purdom, C.E., Hardiman, P.A., Bye, V.J., Eno, N.C., Tyler, C.R., and Sumpter, J.P. (1994). Estrogenic effects of effluents from sewage treatment works. Chemical Ecology 8, 274–285. Quinsey, P.M., Donohue, D.C., and Ahokas, J.T. (1995). Persistence of organochlorines in breast milk of women in Victoria, Australia. Food and Chemical Toxicology 33(1), 49–56. Raven, P. H. and Johnson, G. B. (1999). Biology. 5th ed. WCB/McGraw-Hill; Boston. Reijinders, P.J.H. (1986). Reproductive failure in common seals feeding on fish from polluted coastal waters. Nature 324, 456–457. Ries, L.A.G., Hankey, B.F., and Miller, B.A. (1991). Cancer statistics review 1973–88. National Institutes of Health, Publication no. 91-2789. US Government Printing Office; Washington, DC. Rogan, W. (1982). PCBs and Cola-colored babies: Japan, 1968, and Taiwan, 1979. Teratology 26, 259– 261. Routledge, E. J., Sheahan, D., Desbrow, C., Brighty, G., Waldock, M., and Sumpter, J. P. (1998). Identification of estrogenic chemicals in STW effluent. 2. In vivo responses in trout and roach. Environmental Science and Technology 32, 1559–1565. Salazar, M.H. and Salazar, S.M. (1991). Assessing sitespecific effecs of TBT contamination with mussel growth rates. Marine Environmental Research 32, 131–150. Schade, G. and Heinzow, B. (1998). Organochlorine pesticides and polychlorinated biphenyls in human milk of mothers living in northern Germany: Current extent of contamination, time trend from 1986 to 1997 and factors that influence the levels of contamination. Science of the Total Environment 215, 31–39. Siyali, D.S. (1973). Polychlorinated biphenyls, hexachlorobenzene, and other organochlorine pesticides in human milk. Medical Journal of Australia 2, 815–818.

Endocrine Disrupting Chemicals. What? Where?

Society of Environmental Toxicology and Chemistry (SETAC). (2000). Endocrine disruptors and modulators. SETAC Technical Issue Paper. Pensacola, FL. Sonnenschein, C. and Soto, A.M. (1998). An updated review of environmental estrogen and androgen mimics and antagonists. Journal of Steroid Biochemistry and Molecular Biology 65(1–6), 143–150. Stacey, C.I., Perriman, W.S., and Whitney, S. (1985). Organochlorine pesticide residue levels in human milk: Western Australia, 1979–1980. Archives of Environmental Health 40(2), 102–108. Sumpter, J.P. (1998). Xenoendocrine disrupters− environmental impacts. Toxicology Letters 102/103, 337–342. Tabata, A., Kashiwa, S., Ohnishi, Y., Ishikawa, H., Miyamoto, N., Itoh, M., and Magara, Y. (2001). Estrogenic influence of estradiol-17β, p-nonylphenol and bisphenol A on Japanese Medaka (Oryzias Iatipes) at detected environmental concentrations. Water Science and Technology 43(2), 109–116. Tanabe, S., and Kunisue, T. (2007). Persistent organic pollutants in human breast milk from Asian countries. Environmental Pollution 146, 400–413. Tyler, C.R. and Routledge, E.J. (1998). Oestrogenic effects in fish in English rivers with evidence of their causation. Pure and Applied Chemistry 70(9), 1795–1804.

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United States Environmental Protection Agency (U.S. EPA). (1997). Special report on environmental endocrine disruption: An effects assessment and analysis. EPA/630/R-96/012, February, 1997. U.S. Environmental Protection Agency; Washington, DC. vom Saal, F.S., Timms, B.G., Montano, M.M., Palanza, P., Thayer, K.A., Nagel, S.C., Dhar, M.D., Ganam, V.K., Parmigiani, S., and Welshons, W.V. (1997). Prostate enlargement in mice due to low doses of estradiol or diethylstilbestrol and opposite effects at high doses. Proceedings of the National Academy of Sciences of the United States of America 94, 2056–2061. Weis, S., Weis, P., and Wang, F. (1987). Developmental effects of tributyltin on the fiddler crab, Uca pugilator, and the Killifish, Fundulus heteroclitus. In: Proceedings of the Organotin Symposium of the Oceans ’87 Conference, Halifax, Canada. pp. 1456– 1460. IEEE, Piscataway, NJ, USA. Wolff, M.S., Toniolo, P.G., Lee, E.W., Rivera, M., and Dubin, N. (1993). Blood levels of organochlorine residues and risk of breast cancer. Journal of National Cancer Institute 85, 648–662. Yu, M., Hsu, C., Guo, Y., Lai, T., Chen, S., and Luo, J. (1994). Disordered behaviour in the early-born Taiwan Yucheng children. Chemosphere 29(9–11), 2413–2422.

Chapter 2 Analysis of PCBs in Food Manuela Melis and Ettore Zuccato

Introduction Polychlorinated biphenyls (PCBs) are apolar and nonflammable industrial fluids characterized by high electrical isolation properties and good thermal and chemical stability. These chemicals were intentionally produced as technical mixtures and are widely used in industrial and commercial applications as dielectric fluids, organic diluents, plasticizers, adhesives, and flame retardants, especially during the 1970s. Even though their production and use has been banned for a few decades in the United States and Europe, they are still widespread pollutants in air, soil, sediments, and biota, especially in industrialized regions. In addition, PCBs can be unintentionally produced as by-products of some chemical processes or degradation of chlorinated organic compounds. Chemically, the term PCBs includes a family of 209 different congeners that can be divided into two principal groups according to their toxicological properties. The group of dioxin-like PCBs (DL-PCBs), including non-ortho and monoortho PCBs and consisting of 12 congeners, shows toxicological properties similar to those of dioxins. The other group, non-dioxinlike PCBs (NDL-PCBs), includes the most abundant congeners in the environment and human tissue; those toxicological characteristics have not been completely clarified. Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

PCBs are lipophilic and bioaccumulate in the food chain. The diet, in particular the consumption of fish and animal products, is the major route of exposure for humans. After intake, due to their hydrophobic characteristics and resistance to metabolic degradation, these substances can accumulate in fatty tissues, where they can exist as mixtures of different congeners with half-lives of several years. Because DL-PCBs can adopt a coplanar conformation and become isosteric with dioxins, these congeners can bind the aryl hydrocarbon receptor (AhR) and have toxicological characteristics similar to those of dioxins (Barouki et al. 2007). NDL-PCBs, indeed, show a different toxicological profile, and some studies suggest that these congeners can reduce dopamine neurotransmitter levels, and as a result, interfere with calcium homeostasis in neurons (Brown et al. 1998; Seegal 1998; Tilson et al. 1998).

Analysis of PCBs in food Because of their physicochemical properties, PCBs can persist in the environment and bioaccumulate in foods. The main source of nonoccupational exposure to these substances for humans is the diet, and this makes it important to analyze food items to estimate the real exposure of the population to PCBs. These compounds are lipophilic and apolar. As a consequence, they have low water solubility, and their highest concentrations are found in fatty foods such as fish and meat rather than in vegetables, cereals, and fruits. Owing to 19

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Analysis of Endocrine Disrupting Compounds in Food

the variety and the complexity of the sample matrix and the possibility of crosscontamination, the analysis of PCBs in foods is difficult (Carabias-Martínez et al. 2005). For instance, because in the 1970s–1980s the use of PCBs in industrial fields was common, these substances may be present in laboratories built during these years. In some cases, important sources of contamination were found in some electronic equipment placed next to the laboratories (Weistrand et al. 1992). After that time, to prevent significant contamination, samples were not allowed to be exposed to the laboratory air for extended periods (Alcock et al. 1994), and some researchers suggested that the air of the laboratory be prefiltered through HEPA and carbon filters (Muir and Sverko 2006). Moreover, plastic materials should be avoided during storage, extraction, and cleanup of samples because such materials may contain plasticizers such as phthalates that might interfere with the target compound in the analysis (de Boer 1999). The procedure for PCB analysis in food consists of several different steps: matrix preparation, extraction from the matrix (often together with other lipophilic matrix components), cleanup or purification, and instrumental determination (Bucholski et al. 1996; Letellier and Budzinski 1999).

Sample pretreatment European Commission (2006) directive 2006/1883/EC of 19 December 2006 established that samples must be stored and transported in glass, aluminum, polypropylene, or polyethylene containers, and storage and transportation must be performed in a way that maintains the integrity of the food sample. A basic requirement for a laboratory dedicated to PCB analysis is the availability of sufficient freezer and refrigerator capacity for sample storage and archiving (Muir and Sverko 2006). Samples of tissues are, in fact, usually preserved by freezing after dissection

in small pieces (Hess et al. 1995) and homogenization or grinding to cause rupture of membranes (Sparr Eskillson and Björklund 2000). Storage of wet samples at −25°C is a safe procedure for fish for a period of up to 2 years, and storage of freeze-dried fish samples in a dark place at ambient temperature is also possible. For storage periods longer than 2 years, it is recommended that the samples be stored at −70°C or lower (de Boer 1999); however, dried products are more stable and can be more conveniently stored than wet food samples (Fernandes et al. 2004). Drying with anhydrous sodium sulfate is convenient, particularly for samples with smaller sizes and more lipid-rich matrices, such as oily fish (Fernandes et al. 2004). Drying is particularly important when using nonpolar solvents for solid sample extraction, and the use of sodium sulfate, diatomaceous earth, or cellulose can improve the efficiency of extraction that could be compromised by moisture (Ridgway et al. 2007). Witczak and Chlewinska (2008) have compared the effect of freeze-drying and anhydrous sodium sulfate in the muscles of selected fish species. In fish tissues, freezedrying caused the loss of PCBs during sublimation, especially lighter PCBs (Juan et al. 1999), but the difference in recovery between this method and drying with anhydrous sodium sulfate was not statistically significant. Despite smaller recoveries, freezedrying is recommended for extraction of fish tissues because it saves on solvent and the extracts are easier to prepare. Animal or fish tissue handling can cause problems, however; drying before extraction may not always be practical because these samples can be characterized by high proportions of fats or because cell collapse can cause retaining of the analytes. Tissue matrices, in particular, tend to clump, preventing the extraction by solvent (Ridgway et al. 2007). Dispersion of tissue with solid support can be used to avoid aggregation of sample particles and to enhance solvent extraction (Ridgway et al. 2007).

Analysis of PCBs in Food

Vegetables usually have high water content and should be dehydrated prior to the analysis unless extracted immediately after sampling. Vegetable matter is therefore crushed, chopped, and freeze-dried, or gently dried at 40°–50°C prior to storage and analysis (Liem 1999a). Analysis of solid and semisolid foods are at a disadvantage because liquid samples require fewer pretreatment steps (Carabias-Martínez et al. 2005). Liquid samples are usually freeze-dried for storage (Thomsen et al. 2002). In general, analysis of halogenated contaminants in water is recommended immediately after sampling (de Boer 1999).

Extraction The extraction step serves to isolate analytes from potentially interfering sample components while getting these analytes into a form suitable for analysis (Raynie 2006). Extraction is a fundamental step that prepares food samples for instrumental analysis and consists of a transfer by partition of the analytes from food matrices to an extraction matrix with a simultaneous elimination of interfering substances. PCBs are lipophilic, thus extraction methods are based on the isolation of the lipid fraction from the sample matrix. Methods for the isolation of the fat fraction from individual food differ depending on the type of sample (Liem 1999a). Butter, fats, and oils do not normally require extraction procedures (Cencicˇ Kodba and Brodnjak Voncˇina 2007). Sample weight used for the extraction must be sufficient to fulfill the requirements with respect to sensitivity. There are many satisfactory specific sample extraction procedures that may be used for the products under consideration. Every laboratory may use their preferred procedure as long as it is validated according to internationally accepted guidelines (EU Commission Directive 2006/1883/EC). Below, we describe the most common techniques used for PCB extraction from foods (see also Table 2.1, which sum-

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marizes the principal characteristics of each technique). Liquid–liquid extraction (LLE) Liquid–liquid extraction (LLE) is easy and popular, and it is one of the most common methods of extraction, especially for organic analytes from liquid matrices. Analytes in solution or liquid samples can be extracted by direct partitioning with immiscible solvents mixed by shaking. Choosing the proper solvent polarity for solubilizing the analytes is fundamental, and generally a combination of nonpolar solvents with solvents of various polarity is used. As an example, to extract the lipid fraction of butter for PCB analysis, Ramos et al.(1999) used a mixture of acetone/ hexane (2 :1 v/v), and Battu et al.(2004) used acetonitrile saturated with hexane, then partitioned the acetonitrile fraction with dichloromethane. However, this technique can also be adopted to extract solid samples after homogenization. For instance Mondon et al. (2001) used acetone/hexane (1 : 1 v/v) to extract PCBs from 2 g of sand flathead and Pacific oysters after homogenization. Johansen et al. (1993) extracted 10–20 g of crab tissue with cyclohexane, acetone, and water, and Matthews et al. (2008) used two different methods, dichloromethane (DCM)/cyclohexane (1 : 1 v/v) and hexane/acetone (1 : 1 v/v), to extract PCBs from skin and muscle of edible fish, crustaceans, and shellfish from Queensland, Australia. Zuccato et al. (2004) extracted 25 g of homogenized salmon with 100 mL of 25% hydrochloric acid and 200 mL of 1 : 1 DCM/hexane to study PCB concentrations in salmon samples from Europe. Zuccato and colleagues (2008) used acetone to extract cabbage and concentrated sulfuric acid and DCM/hexane 1 : 1 to extract butter to study PCBs in foods from Belgium, Italy, Portugal, and Spain. This technique is easy but presents several disadvantages, such as the use of large volumes of solvents that have to be

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Large volume

Moderate

Easy to perform

The extract must be evaporated Large volumes of toxic solvents Expensive Time consuming.

Sample size

Solvent consumption

Investment

Advantages

Disadvantages

Long extraction time Large solvent use Cleanup is required The extract must be evaporated One run not enough Water must be reduced before extraction.

No filtration after extraction Reduced consumption of solvents Faster and less expensive than L-L extraction.

Low

50–200 mL

1–30 g

Many hours

Soxhlet Sample is placed in a glass thimble and repeatedly percolated with condensed vapors of the solvent.

Possible retention of lipid fractions The sample must be homogeneous.

It is an extraction technique or a cleanup method No emulsion formation Parallel extraction of many samples Low solvent consumption.

Low

Low volume

Moderate

Fast

SPE Disposable cartridges used to trap the analytes on a solid phase and separate them from the matrix by solvent extraction.

The use of small sample size can reduce the sensitivity of the analysis Fatty matrix requires cleanup steps.

Reduced time and solvent consumption Enhanced extraction efficiency No emulsion formation.

Moderate

Low volume

0.5 g

Moderately fast

MSPD The sample is blended with solid supports with a mortar and pestle. The materials are loaded in syringe barrel and the analytes are solvent extracted.

Filtration and cleanup after extraction Water content must be reduced before extraction.

Speed analysis Modest sample and solvent consumption Parallel extraction of many samples Simple to perform.

Moderate

30–200 mL

1–30 g

10–60 min

Sonication The sample is immersed in an organic solvent in a vessel and placed in an ultrasonic bath at room temperature.

Cleanup may be required Moisture must be controlled Homogenization and drying are required before extraction.

Speed analysis Modest sample and solvent consumption Parallel extraction of many samples. Good results for low-fat sample.

High

Low volume

30 g

Fast

ASE A solid sample is enclosed in a solvent-filled cell and pressurized.

Cleanup and filtration of extracts may be required Moisture must be controlled. Polar solvents are needed.

Speed analysis Modest sample and solvent consumption Parallel extraction of many samples.

High

10–30 mL

0.5–10 g

1–10 min

MAE The solvent of extraction is heated in a vessel in contact with the sample using microwave energy in a vessel.

May require cleanup and filtration of the extracts Moisture must be controlled. Lack of robustness and of universal methods of analysis.

Speed analysis Modest sample and solvent size Parallel extraction of many samples.

High

Low volume

1–5 g

10–60 min

SFE The sample is loaded in a high-pressure vessel and extracted with a SF. Then the analytes are collected in a small volume of solvent or by a cartridge.

L–L, liquid–liquid extraction; SPE, solid-phase extraction; MSPD, matrix solid-phase dispersion; ASE, accelerated solvent extraction; MAE, Microwave-assisted extraction; SFE, supercritical fluid extraction

Moderately long

Large

Extraction time

Analytes in solution or liquid samples can be extracted by direct partitioning with immiscible solvents mixed by shaking.

Description

L–L

Table 2.1. Summary of the extraction techniques used for PCB analysis.

Analysis of PCBs in Food

evaporated after extraction. It is expensive and time consuming and requires the use of highly flammable and toxic solvents. Afterward, it is important to note that in order to apply LLE, the size of the sample should be relatively large (Hess et al. 1995; Bucholski et al. 1996; Carro et al. 2002; Ahmed 2003; Focant et al. 2004a; Ridgway et al. 2007; Beyer and Biziuk 2008). Soxhlet extraction The traditional extraction method for the determination of a wide variety of compounds in food samples is Soxhlet extraction (Carabias-Martínez et al. 2005). Before extraction, animal and fish tissues are macerated and then ground with sodium sulfate and silica; this first passage can reduce the water content, enhancing the extraction efficiency (Hess et al. 1995). In a conventional Soxhlet apparatus, up to 10 g of sample is placed in a thimble holder, which is gradually filled with 50–200 mL of condensed fresh solvent from a distillation flask. When the liquid reaches the overflow level, a siphon aspirates the solute from the thimble holder and unloads it back into the distillation flask, carrying the extracted analytes into bulk liquid. This operation is repeated until extraction is complete (Dean and Xiong 2000; Ridgway et al. 2007). No filtration is required after a leaching step (Luque de Castro and Gárcia-Aruso 1998; Letelier and Budzinski 1999; Carro et al. 2002). Nonpolar solvents, such as n-hexane, have been frequently used for PCB analysis (Hess et al. 1995). Nonpolar solvents are too immiscible with water to penetrate the wet material however, thus a medium polarity or a binary solvents mixture is recommended, such as DCM or hexane/acetone (1 : 1 v/v) (de Boer 1988; Folch et al. 1996). During the last decade, Soxhlet extraction has been used for determination of PCBs in fish, mammals, and mussel tissues (Vives and Grimalt 2002; Manirakiza et al. 2002; Corsolini et al. 2002; Kirivanta et al. 2003; Malavia et al. 2004; Asmund et al. 2004; Isosaari et al. 2006; Pan

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et al. 2007; Koistinen et al. 2008; Witczak and Chlewinska 2008). Zuccato et al. (1999) used Soxhlet to extract (for 8 hours with hexane/ acetone, 9 : 1 v/v) 5 g of lyophilized sample of mixed meat, dairy products, fish, oils, green and other leafy vegetables, cereals, sweets and sweeteners, fruits, alcoholic and nonalcoholic beverages, in order to study the source and toxicity of PCBs in the Italian diet. Focant et al. (2002) extracted 10 g of powdered milk with Soxhlet using pentane/DCM (1 : 1 v/v) as the solvent mixture. Zhao et al. (2009) have homogenated and individually dried vegetables, rice, pulses, pork, and chicken and fish muscle samples that were then Soxhlet extracted with hexane/acetone (3 : 1 v/v). Grassi et al. (2008) have used Soxhlet to extract vegetables with a solution of hexane/ acetone (9 : 1 v/v) for 24 hours. Huckins et al. (1990) have proposed the use of semipermeable membrane during extraction for separating the component fraction from the lipid matrix of foodstuffs. During the extraction, PCBs are allowed to cross the semipermeable membrane of a polyethylene bag, and the lipid matrix is retained; extraction time is shorter than normal, and it is sufficient for a quantitative recovery of all the components of interest. Soxhlet extraction was born from the need to reduce the consumption of solvents, and it is faster and less expensive than LLE. Despite these advantages, it is necessary to evaporate the solvents to concentrate the samples, and a single sample run requires many hours to be completed (Liem 1999b; Abrha and Raghavan 2000; Buldini et al. 2002; Beyer and Biziuk 2008). Moreover, the temperature of the system remains high because samples are extracted by keeping the solvent at the boiling point for a long time, thus the possibility of thermal decomposition of thermolabile analytes cannot be excluded. Solid-phase extraction Solid-phase extraction (SPE) is widely accepted as an alternative to laborious and

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Analysis of Endocrine Disrupting Compounds in Food

time-consuming liquid–liquid methods for extraction of nonvolatile organic compounds such as PCBs from liquid matrices. SPE can be used directly as an extraction technique for liquid matrices or as a cleanup method after solvent extraction (Hennion 1999; Ahmed 2001; Focant et al. 2004a; Gilbert-López et al. 2009). SPE is based on the use of disposable cartridges to trap the analytes and separate them from the matrix. The use of SPE requires that the sample must be homogeneous and in a liquid state prior to addition to a SPE column or disk device. The presence of particulate can complicate the use of SPE, impeding and blocking the flow and creating variability in the particulate content of samples, possibly leading to variability of recoveries (Barker 2000a). The extraction is performed in four steps: conditioning of the SPE column (the functional groups of the sorbent are solvated in order to activate them to permit the interaction with analytes), retention (solutions are passed through the column and the analytes are bound to the bed surface), selective washing (to eliminate the undesired molecules), and elution (the analytes are desorbed and collected for analysis). When a sample solution passes through the activated sorbent bed, analytes are concentrated on the surface while other components pass through the bed. Selection of the appropriate SPE sorbent depends on the mechanism of interaction between the sorbent and the analytes of interest. Many types of sorbent, such as alumina, magnesium silicate, and graphitized carbon are available. Silica is widely used because it is reactive enough to permit its surface to be modified by chemical reaction, yet stable enough to allow its use with a wide range of solvents (Buldini et al. 2002). Nonpolar sorbents can be made of functional groups (C8, C18, cyano, amino, sulfunic acid, etc.) bound to the surface of silica to alter its retention properties or they may be made of functionalized styrenedivinylbenzene synthetic polymers (Focant et

al. 2004a). In particular, C18 cartridges have nonpolar characteristics that retain organochlorine compounds and have a size exclusion function suitable to eliminate several macromolecules coextracted with PCBs. For these reasons C18 cartridges can be used either for extraction or for sample cleanup (Covaci and Schepens 2001). For instance, C18 cartridges have been used for the analysis of PCBs in milk (Picó et al. 1995; Ahmed 2003; Centi et al. 2007), and Dong et al. (2000) and Sun et al. (2002) have used these cartridges to analyze PCBs in water samples from the Yangtze River in China. Diatomaceous earth (diatomite, Extrelut), a form of silica composed of the siliceous shells of unicellular aquatic plants, has also been used frequently for extraction of PCBs. SPE decreases the sample-preparation time and the consumption of solvents, permits the simultaneous removal of several interfering substances, and allows multiple samples to be treated in parallel using small volumes of solvents. SPE is an advantageous technique because there is no emulsion formation and no requirement for repeated extraction or centrifugation steps. However, the presence of particulate in unfiltered samples might cause clogging of the SPE cartridge, which may lead to longer extraction time (Hennion 1999; Ahmed 2001; Björklund et al. 2002; Buldini et al. 2002). Moreover, during extraction by a SPE method, part of the lipids may remain in the cartridge, even after elution with nonpolar solvents (Focant et al. 2004a). To solve this problem Sjödin et al. (2004) used a solidphase dispersion of diatomaceous earth in the SPE cartridge to determine PCBs. This permitted the extraction of milk without the inconvenience of loosing part of the lipids. Matrix solid-phase dispersion Matrix solid-phase dispersion (MSPD), a tissue disruption/extraction method, uses a mortar and pestle to blend octadecylsilylderivatized silica (C18) or other chemically

Analysis of PCBs in Food

modified solid supports with the tissue sample (Crouch and Barker 1997). For extraction of analytes from animal tissues, C18-bonded silica supports are frequently used, and liquid samples can be dispersed on Florisil or C18bonded silica. Ramos et al. (2004) have used modified silica treated with sulfuric acid (44% w/w) for fat removal and simultaneous selective MSPD of PCBs from pork tissue samples. The particle size is important, and most applications have reported using a 40μm diameter particle. The mortar and pestle used should be made of glass or agate, because porous materials and porcelain have been shown to lead to analyte and sample loss. The analysis of food by MSPD requires, in general, a small sample size (0.5 g) blended in a mortar with 2 g of bonded-phase solid support, typically C18 or C8. When MSPD blending is completed, the material is transferred in a syringe barrel that represents the column or some other appropriate device. A second frit or plug is often placed at the top of the sample before it is compressed with a plunger. Larger glass columns have also been used for applications involving large sample amounts, such as to extract traces of PCBs from milk (Ramos et al. 1999, 2004). When the sample has been loaded, it is compressed to form a column packing, and the elution of the column is made by addition of the solvent. There are two possibilities: (1) target analytes are retained on the column and interfering compounds are eluted in a wash step, and the analytes are subsequently eluted with a different solvent, or (2) interfering compounds are selectivity retained on the column and the target analytes are eluted directly. Crouch and Barker (1997) have observed that a MSPD column can provide a separation of lipids and other nonpolar materials from PCBs present in the sample if the elution solvent chosen has the correct solvent strength in relation to matrix and column support. The elution is usually conducted by gravity flow, but it is possible to apply vacuum to the bottom or pressure to the head of the column (Barker

25

2000a, b; Kristenson et al. 2006; Bogialli and Di Corcia 2007). Some MSPD extracts are clean enough to be subjected to instrumental analysis directly, but a cleanup step is frequently required, especially for fatty matrices. For this purpose, co-columns have been used to obtain further fractionation and to assist in extraction cleanup. A co-column material (such as Florisil, alumina, or silica) can be packed in the bottom of the same cartridge containing the matrix blend or used as an external column that collects and fractionates the sample that emerges from the MSPD column (Bogialli and Di Corcia 2007; Ridgway et al. 2007). The application of MSPD for the analysis of foods is therefore based on the blending of a viscous, solid, or semisolid sample with an abrasive solid support material (that has been derivatized) to produce bound with organic groups on its surface, such as silica-based SPE materials. MSPD, by using abrasive materials and shearing forces generated by the blending process, permits disruption of the architecture of the sample, breaking its components into smaller pieces. Unlike SPE, in the MSPD technique the sample is dispersed through the column, becoming part of the system. This technique has been applied to the analysis of PCBs in animal tissues (fish, oysters, bovine, ovine, etc.), milk, and eggs. The use of MSPD reduces time of analysis and solvent use, eliminates emulsion formation, and enhances extraction efficiency because the entire sample is exposed to the extractant. The disadvantage of this technique is the small size of the sample, which can reduce sensitivity, and the lack of homogeneity (Kristenson et al. 2006; Bogialli and Di Corcia 2007). Valsamaki et al. (2006) have used MSPD to measure PCBs in chicken eggs. They used Florisil as dispersion sorbent and 10 mL of DCM/hexane (1 : 1 v/v) as solvent for extraction. A one-step extraction–purification method for the determination of PCBs in fat tissues was developed by Ramil Criado et al.

26

Analysis of Endocrine Disrupting Compounds in Food

(2004), who used MSPD with different combinations of phase sorbent and elution solvents to extract PCBs from butter, chicken, and beef fat.

al. (2001) used this technique with cyclohexane and acetone to extract the lipidic fraction from top predator of the Barens Sea. Accelerated solvent extraction

Ultrasonic-assisted extraction Ultrasonic-assisted extraction is a simple technique in which the sample is immersed in an organic solvent in a vessel and placed in an ultrasonic bath at room temperature; sonication involves the use of sound waves to stir the sample immersed in the organic solvent. Energy, in the form of acoustic sound waves in the ultrasound region above 20 kHz, is used to accelerate mass transport and mechanical removal of analytes from a solid matrix by a process called cavitation; the formation and implosion of vacuum bubbles through the solvent induce a greater direct contact of solvent into the solid matrix and improve the sample extraction efficiency (Fidalgo-Used et al. 2007). Apart from the polarity of the solvent, the efficiency of the extraction is dependent on the homogeneity of the matrix (Hess et al. 1995) and by the presence of water, which can reduce the efficiency of the extraction (Ridgway et al. 2007). The time to extract a sample of 1–30 g with 30–200 mL of solvent is 10–60 min for one cycle, and repeated extraction may be required (Sparr Eskillson and Björklund 2000). Ultrasonicassisted extraction has been recently carried out using a dynamic extraction setup that continuously supplies extraction solvent to the extraction vessel. According to Priego-Capote and Luque de Castro (2004), ultrasonicassisted extraction is characterized by high speed of analysis and limited solvent and sample consumption. On the contrary, however, Beyer et al. (2008) and Sparr Eskilsson and Björklund (2000) reported that this technique requires long extraction time, repeated extractions, and large volumes of solvents, whereas according to Fidalgo-Used et al. (2007), it also requires filtration of the extract after extraction. To study organochlorines in crustaceans and fish species, Borgå et

Accelerated solvent extraction (ASE) is also known as pressurized fluid extraction (PFE) or pressurized liquid extraction (PLE). The basic experimental setup using the Dionex ASE 200 accelerated solvent extractor (Dean and Xiong 2000; Focant et al. 2004a) permits the automation of the process and the extraction of a large number of samples sequentially (Gilbert-López et al. 2009). ASE can be considered a new version of a Soxhlet apparatus that can operate at high temperature and pressure (Mendiola et al. 2007). A solid sample, up to 30 g in weight (Dean and Xiong 2000), is enclosed in a cartridge that is filled with an extraction solvent or solvents mixture. For extraction of PCBs from environmental samples, usually hexane/acetone (1 : 1 v/v) or acetone/DCM (1 : 1 v/v) (Dean and Xiong 2000; Sparr Eskilsson and Björklund 2000) are used. In general, appropriate solvent mixtures for extraction are chosen according to their physicochemical properties, such as boiling point, specific density, and toxicity. Amounts of solvent consumed will depend on the number of extraction cycles. For instance, to extract PCBs from fish samples, two cycles are considered to be best for optimizing time savings and solvent consumption (Suchan et al. 2004). Especially when a nonpolar solvent is used, drying the sample before extraction is a fundamental step because moisture can reduce extraction efficiency (Gilbert-López et al. 2009). Generally, for biological samples, drying of the matrix is carried out before ASE (Ramos et al. 2002; Wiberg et al. 2007). However, a desiccant such as sodium sulfate, diatomaceous earth, or cellulose can also be added directly to the extraction cell, together with sorbent materials that can be used to provide in situ cleanup, when separation of PCBs from coextracted lipids is needed

Analysis of PCBs in Food

(Suchan et al. 2004). Sometimes, sodium sulfate would not be sufficient to completely dehydrate biological samples, particularly when they contain large volumes of water. In this case, hydromatrix consisting of freeflowing granules can help by dehydrating the sample directly in the cell (Saito et al. 2004). The use of more polar solvent (acetonitrile, methanol, ethyl acetate, etc.) or a polar solvents mixture (hexane/acetone, hexane/ acetonitrile, etc.) can also improve the extraction of wet samples, thus reducing the need for drying steps (Focant et al. 2004a; Carabias-Martínez et al. 2005). To purify samples during ASE, Müller et al. (2001) proposed in-cell fat removal by packing the sample dispersed in a sandNa2SO4 mixture (1 : 1 w/w) on top of a multilayer column containing acid silica and neutral silica to remove sulfuric acid traces from the n-hexane eluate. This technique provided satisfactory results for samples with low fat, such as cereals-based foodstuff, but also for more fatty materials, such as powdered milk. Björklund et al. (2001) have studied five types of fat retainers for the lipidfree extraction of PCBs from fat-containing matrices (lard, cod liver oil, and fish meal) using ASE: Florisil, acid alumina, neutral alumina, basic alumina, and sulfuric acidimpregnated silica. Florisil and sulfuric acidimpregnated silica provided the best results. With ASE the sample is extracted at high pressure (500–3000 psi) and temperatures (50°–200°C) above the boiling point of the solvents for a short time (5–10 min). The higher temperature increases the ability of the solvent to solubilize the analytes, disrupting the strong interaction between solute molecules and matrix (Suchan et al. 2004); decreases the viscosity of the solvents, allowing a better penetration into the matrix; and enhances the extraction efficiency (Richter et al. 1996; Buldini et al. 2002; Fidalgo-Used et al. 2007). For fish homogenate samples, optimal PCB extraction recoveries were observed in the temperature range of 90°– 120°C. The combination of high pressure and

27

temperature allows rapid extraction with little solvent consumption but requires the use of very expensive and specialized equipment (Liem 1999b; Carro et al. 2002; Björklund et al. 2006; Mendiola et al. 2007; Beyer and Biziuk 2008). Compressed gas is then used to purge the sample extract from the cell to the collection vessel (Richter et al. 1996). PCB analysis in oyster tissues has been done using isooctane (Richter et al. 1996) and pentane (Gómez-Ariza et al. 2002). ASE has been used for the analysis of PCBs in mussels (Schantz et al. 1997; Gómez-Ariza et al. 2002), fish tissues (Schantz et al. 1997; Gómez-Ariza et al. 2002; Kitamura et al. 2004; Suchan et al. 2004; Jiang et al. 2007), and meat samples (Kitamura et al. 2004; Saito et al. 2004). Focant et al. (2001) extracted freeze-dried poultry and mackerel (fillet) by PLE to measure coplanar PCB concentrations. Microwave-assisted extraction Microwave-assisted extraction (MAE) consists of using microwave energy to heat the extraction solvent in contact with the sample. Microwave energy causes molecular motion by migration of ions and rotation of dipoles, and at the frequency used in commercial systems, usually 2450 MHz, the heating of the molecules is rapid. The effect of microwave energy is strongly dependent on the nature of the matrix and solvent extraction, and MAE has been successfully applied to the extraction of solid and semisolid matrices (Gilbert-López et al. 2009). The water content of the matrix is very important because water molecules have a high dipole moment and, as a consequence, adsorb microwave energy strongly, leading to efficient heating of the sample (Camel 2000). Hummert et al. (1996) reported that hexane is good to use to extract PCBs from fatty animal tissues using MAE. When the analytes can be degraded by high temperature, it is advisable to use a solvent with a low dielectric constant. Thus it is common practice to use a

28

Analysis of Endocrine Disrupting Compounds in Food

binary mixture (e.g., hexane/acetone, 1 : 1 v/v) where only one of the solvents absorbs microwaves (Fidalgo-Used et al. 2007). The amount of solvent needed for a single sample is in a range of 10–30 mL; the volume of solvent must be sufficient to ensure that the entire sample is immersed (Sparr Eskilsson and Björklund 2000). This technique can be performed using two technologies: a closed vessel (PMAE) under controlled pressure and temperature or an open vessel (FMAE) under atmospheric pressure. In the closed vessel system, the solvent can be heated above its boiling point at atmospheric pressure. These conditions enhance the efficiency of extraction, permit the control of temperature during the extraction, and reduce the time of extraction. The differential temperature between solvent and sample facilitates the transport of the analyte from the sample to the solvent. Several samples can be simultaneously extracted (Smith 2003; Focant et al. 2004a; Gilbert-López et al. 2009). Usually, sample size ranges from 0.5 to 10 g, and 10 mL of solvent is sufficient for a closed-vessel extraction requiring 1 to 10 min to be completed (Letelier and Budzinski 1999; Camel 2000; Buldini et al. 2002). In the open system, the maximum temperature is determinated by the boiling point of the solvent at atmospheric pressure (Camel 2000). MAE offers savings in time and solvent consumption (Xiong et al. 2000), it is cheaper than ASE and supercritical fluid extraction (SFE; Ahmed 2003), and it is a good alternative to Soxhlet because a good extraction efficiency can be achieved with less solvent and a shorter extraction time (Fidalgo-Used et al. 2007; Ridgway et al. 2007). A disadvantage of this method is that extracts must be filtered after extraction, polar solvents are needed for extraction, and equipment is expensive (Fidalgo-Used et al. 2007; Beyer and Biziuk 2008). Probably for these reasons MAE has been rarely used for PCB extraction (Björklund et al. 2002). For example, Carro et al. (2000) have used closedvessel MAE to extract PCBs in the marine

mussel. MAE, however, does not allow separation of the analytes from other interferents; thus, it is desirable to eliminate these compounds after extraction using appropriate cleanup steps (Letelier and Budzinski 1999; Carro et al. 2000). Supercritical fluid extraction A supercritical fluid (SF) is defined as an element or compound above its critical pressure and temperature (Smith 1999). SFs have a density similar to liquids but lower viscosity. During extraction, a SF diffuses through solid matrices like a gas, but it dissolves analytes like a liquid (Buldini et al. 2002). This combination of properties results in a SF that has higher solvating power and may extract analytes faster and more efficiently than do liquids. Moreover, the density of the SF may be adjusted by varying temperature and pressure, thus enhancing efficiency and selectivity of the extraction and reducing thermal degradation (Camel 2001; Buldini et al. 2002; Zougagh et al. 2004; Fidalgo-Used et al. 2007). Several solvents can be used as SF, such as carbon dioxide (CO2), nitrous oxide, ethane, propane, n-pentane, ammonia, etc. (Zougagh et al. 2004). CO2 is currently the solvent of choice because it reaches the supercritical state at a relatively low pressure (7 MPa) and low temperature (31.3°C) and has low toxicity. It is also nonflammable, noncorrosive, chemically inert, and it has low cost (Luque de Castro and Jimenez-Carmona 2000; Buldini et al. 2002; Smith 2003; Zougagh et al. 2004; Fidalgo-Used et al. 2007). Nitrous oxide also is a good supercritical fluid (but it is very flammable and potentially dangerous because of its oxidizing power) (Raynie 1993; Mendiola et al. 2007), whereas ammonia is too corrosive and reactive (Luque de Castro and Jimenez-Carmona 2000; Buldini et al. 2002; Mendiola et al. 2007). The use of CO2 might be limited by its inadequate solvating power of highly polar

Analysis of PCBs in Food

analytes, as it is a nonpolar solvent. However, its polarity can be adjusted with addition of organic solvents, called modifiers, to enhance its solvating properties (Dean and Xiong 2000; Camel 2001; Zougagh et al. 2004). For instance, addition of 1–10% methanol to CO2 expands its extraction range to include more polar analytes (Buldini et al. 2002; Smith 2003). However, a modifier enhances the polarity of CO2 but can decrease selectivity because impurities may be coextracted with target analytes. Moreover, the presence of a modifier changes the values of critical pressure and temperature. During an extraction cycle, static (fluid immobilized with the sample in the closed vessel) and dynamic (percolation of the fluid through the cell) modes can be used together to ensure both penetration of the matrix by the fluid and to avoid saturation of the fluid (Focant et al. 2004a). All supercritical fluid extraction (SFE) instruments contain six basic components; namely, the supply of high-purity CO2, a supply of high-purity organic modifier, the pumps, the oven for the extraction cell, the pressure outlet or restrictor, and the collection vessel (Dean and Xiong 2000). The extraction vessel is filled with a sample and placed in a heated extraction chamber. The pump is used to supply a known pressure of the SF to an extraction vessel that is heated to a temperature above the critical temperature of the SF. During the extraction, the target analytes are removed from the sample matrix into the fluid and swept into a decompression region. Here, the fluid becomes a gas and is vented, and analytes leaving the gas are collected in a vial that contains a small volume of solvent (so-called solvent mode). When the collecting liquid at the outlet of the extractor is substituted with a sorbent cartridge, such as C18, the mode of extraction is the so-called sorbent mode (Gilbert-López et al. 2009). SFE is a popular method for extraction of analytes from solid matrices, but there are problems with liquids, which need solid

29

support materials. When the matrix is not solid, it must be retained on an appropriate support, such as a natural matrix or a synthetic support, such as glass wool or diatomaceous earth, to ensure an effective attack by the SF (Luque de Castro and JimenezCarmona 2000; Smith 2003). The nature of the matrix and its physical properties are very important for the success of the extraction. One problem encountered in practice is that CO2 is immiscible with water; this makes the extraction of liquids or wet samples difficult. The quantity of food required is 1–5 g, and the time of extraction is 10–60 min (Sparr Eskilsson and Björklund 2000). Solid foods require homogenization without the aid of a liquid, especially water. Grinding of the matrix increases surface area for extraction, but presence of moisture limits the diffusion of the SF inside the matrix (Lehotay 1997; Camel 2001; Buldini et al. 2002). For these reasons an additional step of drying or freezedrying before grinding is required. For liquid food, two strategies have been used: the adsorption of the sample onto porous and inert substrates or the coinjection of the sample with the SF in the extraction vessel (Mendiola et al. 2007). Also, in this case the water content of the matrix must be controlled because water is a strong cosolvent and alters SF strength (Buldini et al. 2002). The use of a drying agent is the most common way to retain water if it was not previously removed from the sample: drying agents can help the extraction and the homogenization of the sample (Lehotay 1997). Antunes et al. (2003) have studied the effect of matrix pretreatments on PCB extraction efficiency with SFE by using three different fish sample preparations: fresh fillet, fresh fillet mixed with anhydrous sodium sulfate, and freeze-dried fillet. SFE did not efficiently extract PCBs from fresh fish, even after anhydrous sodium sulfate addition, whereas in freeze-dried fish, PCB extraction was optimized at 328 K and 22 MPa. Pressure had a significant effect on the extraction

30

Analysis of Endocrine Disrupting Compounds in Food

whereas temperature did not affect significantly the efficiency of extraction. Schantz et al. (1998) compared the extraction efficiency of PCBs by Soxhlet and by SFE using mussel tissue standard reference materials, which are well characterized, homogenous, and widely available. Results were comparable, but SFE had several advantages over Soxhlet, including reduced time of extraction and solvent consumption. Disadvantages of SFE are matrices problems (presence of water), high cost of the instruments, lack of robustness, lack of a universal method that works for all analytes and matrices, and need for cleanup of the sample after extraction (Luque de Castro and JimenezCarmona 2000; Järemo et al. 2000; Camel 2001; Buldini et al. 2002; Carro et al. 2002; Antunes et al. 2003; Zougagh et al. 2004; Gilbert-López et al. 2009). PCBs in fish and seafood have been extracted frequently by SFE using CO2 (Björklund et al. 2002). Järemo et al. (2000) have added basic alumina to selectively extract PCBs from model fat samples (lard fat and phospholipidic mixture obtained from egg yolk) using SFE. Hale and Gaylor (1995) also added alumina directly for PCB extraction from fish tissue and obtained a coextraction of lipid <0.1%. Punín Crespo and Lage Yusti (2005) have used SFE to extract PCBs from seaweed mixed with alumina. Bayarri et al. (2001) have extracted persistent organic pollutants (POPs), including PCBs from freeze-dried edible Adriatic fishes.

sary to separate PCBs from other components as much as possible prior to gas chromatography (GC) analysis in order to limit coelution problems. Cleanup steps minimize matrix effects, improve detector response and reproducibility, and extend the lifetime of the capillary column (Rimkus et al. 1996; de Boer and Law 2003; Agustin et al. 2005; Muir and Sverko 2006). Particularly for highly lipidic samples (>10% lipid), such as some fish tissues (e.g., salmon), a lipid removal step must always be considered after extraction (Muir and Sverko 2006). Methods for cleanup of the sample can be divided into two groups: nondestructive (gel permeation and adsorption columns) and destructive (acid digestion and saponification). Cleanup is a laborious step and may require more than one technique to efficiently remove interfering lipids from the sample. Fractionation consists of making a series of group separations prior to final analysis. The cleaned extract contains organohalogen compounds other than PCBs that need to be separated. Moreover, it is necessary to separate mono-ortho PCBs and non-ortho PCBs into different groups because the range of concentrations of the PCBs is too large for all congeners to be measured in a single step without additional concentration or dilution. Afterward, it is essential to separate DLPCBs from other congeners to assess the toxicity of the PCBs (Hess et al. 1995).

Cleanup and fractionation

Gel-permeation chromatography (GPC), also known as size exclusion chromatography, is an excellent technique for quantitative separation of compounds up to a molecular mass of 400 Da (e.g., organochlorine compounds) from macromolecular compounds (600– 1500 Da) (Tekel and Hatrik 1996). It is generally recommended that extracts obtained from complex matrices with high lipid contents, such as fish (Gilbert-López et al. 2009), be purified. The sample extracted is dissolved

During the extraction step, PCBs move from the food to the extraction matrix with the lipid fraction, along with several coextracted interfering compounds that can be at concentrations of orders of magnitude higher than those of PCBs. After extraction, there is therefore a need for additional steps to purify PCBs from lipids (van Leeuwen and de Boer 2008; Gilbert-López et al. 2009). It is neces-

Gel-permeation chromatography

Analysis of PCBs in Food

in an apolar solvent, such as a mixture of cyclohexane/dichloromethane (1 : 1 v/v) or cyclohexane/ethyl acetate (EtOAc; 1 : 1 v/v), and is injected in a GPC system comprising a liquid chromatography pump, a GPC column, a detector (optional), and a fraction collector (Tekel and Hatrik 1996; GilbertLópez et al. 2009). Very apolar solvents, such as DCM, are then applied as eluent to prevent adsorption of the analytes to the column material. Also, elevated temperatures can influence the adsorption whereas permeation is unaffected. GPC with crosslinked dextran gels has been used to separate molecules in aqueous or buffered solvents on the basis of molecular size and partition and absorption characteristics. The divinylbenzene-linked polystyrene gel (Bio Beads SX-3) is still the most commonly used sorbent for GPC; rigid copolymers (such as P1 gel, Envirogel) allow faster analysis and provide better elution characteristics (Ahmed 2001). GPC on Bio Beads SX-3 can be applied to the analysis of fat to measure organochlorine compounds. Sixty grams of Bio Beads SX-3 can accommodate up to 1 g of fat and achieves the separation of PCBs from fish lipids with a size exclusion of 400 Da (Muir and Sverko 2006). The GPC column, which consists of polymer porous microspheres, retains the molecules that are small enough to enter the pores. Lipid molecules, which have a size of 600–1500 Da, are too large to enter these pores and thus are unretained and eluted first from the column (Tekel and Hatrik 1996; Gilbert-López et al. 2009). However, a single step can be insufficient to completely remove interfering lipids from a sample. In this case, placing a second GPC column in series or using high-quality gels may improve the separation. Minicolumns (silica gel, Florisil) are often used for cleanup (Rimkus et al. 1996; Tekel and Hatrik 1996; de Boer and Law 2003; Gilbert-López et al. 2009; Goñi et al. 2009). GPC can be easily automated by simply incorporating an autosam-

31

pler and an automated fraction collector (Tekel and Hatrik 1996; de Boer and Law 2003; Goñi et al. 2009), and a fractionation time interval can be selected to obtain a clean extract (Gilbert-López et al. 2009). However, GPC does not separate PCBs from interfering compounds of the same molecular mass range, and in this case an additional fractionation is usually required (Smedes and de Boer 1997). Adsorption columns Separation of PCBs from coextracted molecules is simple for low-lipidic samples such as vegetables. Generally, small silica gel or Florisil columns should be sufficient for this purpose. Separation and fractionation of PCBs are achieved by applying the extract in a small volume of apolar solvent and fractionating it by eluting with hexane, followed by one or two other solvents of increasing polarity. Polar compounds are retained on the column, and nonpolar compounds are sequentially eluted (Muir and Sverko 2006). Florisil is one of the oldest materials used to clean up PCBs. Florisil is a mixture of several inorganic oxides with SiO2 and MgO as its main components; the composition varies from batch to batch, and as a consequence, this variability does not contribute to the robustness of the procedure (Smedes and de Boer 1997). In its active form, it retains only planar PCBs, whereas the other PCBs elute almost unretained. However, highly activated Florisil can lose its activity easily. Florisil is particularly indicated for fatty food cleanup (Ahmed 2001). Alumina very efficiently removes fat from the extract, and an alumina adsorption column may be used in combination with, or instead of GPC for this purpose (Hess et al. 1995). Alumina is fully activated when dried at 180°C, and it can retain all PCBs; however, its high activity is sensitive to moisture and decreases rapidly after contact with the sample. As a consequence, the sample

32

Analysis of Endocrine Disrupting Compounds in Food

capacity of activated alumina is relatively low (Hess et al. 1995; Smedes and de Boer 1997). During elution, the retention of PCBs is lower than that of other interfering compounds such as polyaromatic hydrocarbons (PAHs); therefore, alumina is an ideal material for the isolation of PCBs from other interfering compounds (Smedes and de Boer 1997). Alumina, Florisil, and silica have been used in different mesh sizes to separate the coextracted materials from PCBs, and the different sorbents have been used separately or in combination (Hess et al. 1995). Chromatographic columns containing alumina, silica gel, and Florisil, or combination of these adsorbents, cannot always guarantee lipid-free extract, but these techniques are used with success for the analysis of contaminants in several food matrices (Beyer and Biziuk 2008). Activated carbon has also been extensively used to separate non-ortho and mono-ortho PCBs from other PCB congeners. It has high affinity for organics in ultratrace levels, and it is cheap, readily available, and easy to use; however, its impurities make its use problematic (Hess et al. 1995; Liem 1999a). Saponification Lipids can be removed by saponification with small volumes of solvent and 20% ethanolic potassium hydroxide at 70°C for 30 min (Hess et al. 1995). Saponification of fats to their corresponding glycerols and carboxylates facilitates the release of PCBs from a fatty matrix and can also selectively degrade many interfering substances (Xiong et al. 2000; Llompart et al. 2001). Saponification and alkaline decomposition are generally applied to the extract and not directly to the food (Ahmed 2003). However, this treatment can cause a loss of chlorine atoms, especially for more highly chlorinated PCBs, and traditional saponification is a time-consuming technique (de Boer 1999; Carro et al. 2002; Agustin et al. 2005).

Acid digestion Concentrated sulfuric acid is mixed with the lipid extract (dissolved in a proper solvent) and stirred for a few minutes; this step is repeated five to six times. The organic layer is then separated in a funnel, and the solution is evaporated to dryness and redissolved in a proper organic solvent to be analyzed (Ahmed 2003). Alternatively, sulfuric acid is added to the silica in a column and the sample is applied (Beyer and Biziuk 2008). The advantage of this technique is that it is fast, efficient, and can remove large quantities of lipids. A column of 50 g of silica impregnated with sulfuric acid can remove up to 10 g of lipid from the extract (Hess et al. 1995).

Instrumental analysis The complexity of food matrices, taken together with the low concentration of PCBs (picograms per gram), requires highly sensitive and selective analytical techniques (Danielsson et al. 2005; Focant et al. 2005; Dorne et al. 2009). The requirements of analytical methods for official control of PCBs in food are defined in EU Commission Regulation 2006/1883. The EU distinguishes reference methods from screening methods for monitoring the levels of DL-PCBs in foodstuffs. Screening methods are used to detect DL-PCBs at the level of interest and comprise GC-MS and bioassays. These methods have the capacity of a high sample throughput and are used to screen large numbers of samples for positive results. They are designed to avoid false negatives. Confirmatory methods provide full or complementary information, enabling the DL-PCBs to be identified and quantified at the level of interest; these methods comprise high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) methods. The specific quantification limit of an individual congener is the concentration of the analyte in the sample that produces an instrument

Analysis of PCBs in Food

response (for two different monitored ions) with a signal-to-noise ratio (S/N) of 3 : 1 for the less intense signal. In the United States, EPA method 1668B (Environmental Protection Agency, 2008) set the requirements for the determination of DL-PCBs in different matrices, including foodstuffs, by HRGC/HRMS; the 12 dioxinlike congeners are determined by isotope dilution quantitation technique, and other congeners are determined by internal standard quantitation technique. Isotope dilution quantitation technique is generally performed by using stable isotope-labeled (13C12) analogs of the analytes as an internal quantification standard. These labeled compounds (one for each homologous group) are added in known amounts to the sample prior to extraction or cleanup and are assumed to be equal to native analytes with respect to extraction, cleanup, and GC properties. The labeled compounds can be distinguished from the native compounds on the basis of their mass spectral differences and are also used as a guide for identification (Liem 1999a). A blank and a reference sample must be included in each test series, which is extracted and tested at the same time under identical conditions. The reference sample must show a clearly elevated response in comparison to a blank. Gas chromatographic analysis Gas chromatography has always been the method of choice for the determination of PCBs due to the volatility and the complexity involved in separating the isomers of these molecules (de Boer 1999). PCBs are present in a complex mixture of congeners, ranging from monohalogenated to heavily halogenated compounds (Santos and Galceran 2002). PCBs are present in food in ultratrace levels in the presence of much higher levels of other chemical pollutants; as a consequence, highly selective and sensitive analytical methods are required to determine these molecules because, for accurate and

33

reliable data, each congener peak must be resolved from other compounds and should appear as a single peak (Marriot et al. 2003; Haglund et al. 2008). One-dimensional gas chromatography analysis After PCB identification, packed-column GC was the only method of separation available, and total PCB determination (based on comparing an environmental sample with a technical PCB mixture) was all that could be achieved. With the introduction of fused silica capillary columns, the congenerspecific determination of PCBs was possible, but one single (or 1DGC) column GC presents the required resolution only when the number of target analytes is limited. Fused silica open tubular capillary columns, generally coated with nonpolar (phenyl methylpolysiloxane) or medium polarity (cyanopropyl methylpolysiloxane) chemically bonded liquid phases, are almost universally used for GC separation of PCBs, but there is no single capillary column available that can separate all 209 congeners, neither with a length of 50 m nor an inner diameter (I.D.) of only 0.15 mm (Castello and Testini 1996; de Boer 1999; Phillips and Beens 1999; Santos and Galceran 2002; de Boer 2003; Marriott et al. 2003; Miur and Sverko 2006; Korytg´ r et al. 2006). Mullins et al. (1984) have defined the basic technology for high-resolution separation of PCB congeners by using a 5% phenyl methylsilicone phase and a long temperature program (100 min). Congener separation can be improved using a 60 m × 0.25 mm I.D. column with hydrogen carrier gas, which offers a good resolution. Helium may be used as the carrier gas as an alternative to hydrogen, but the I.D. of the column must be >0.20 mm to avoid practical difficulties. The columns used for PCB separation have a length of 30–60 m and a typical film thickness of 0.25 μm. The column diameter is proportional to the resolution, with typical column

34

Analysis of Endocrine Disrupting Compounds in Food

diameters in the range of 0.25 to 0.32 mm, but narrow diameters of 0.10–0.15 mm are also possible. These small dimensions require high gas pressure, up to 150 psi, which can cause problems because not all GC instruments are able to deliver carrier gas at a pressure high enough to achieve an optimum flow rate (de Boer and Law 2003; van Leeuwen and de Boer 2008). Single-column high-resolution GC is normally the final stage of PCB analysis, and the temperature of analysis is in a range from 100°C to 280°–320°C. The selection of GC condition and the phase of the capillary column, along with its physical parameters, is fundamental to obtaining a single peak resulting from the elution of the congeners, free from interference of other PCBs (Hess et al. 1995; Miur and Sverko 2006). For the commonly used capillary columns (DB-1, DB-5, DB-1701, BPX-5, HT-8, SE-54, SIL8, SP-2330, and CP-SIL-9) several authors have reported critical separation of PCBs 28/31, 49/52, 77/110, 101/84, 118/149, 105/153/152. When a single-column is used for complex pollutant mixture analysis, the result might be lack of resolution and identification. For this reason, the trend is toward congener-specific analysis (Liem 1999b; Marriott et al. 2003; Bianco et al. 2008; van Leeuwen and de Boer 2008). Multidimensional gas chromatography: heart-cut MDGC and GCxGC Chromatographic techniques offer highresolution separation, but for complex mixtures the separation power of a single chromatographic procedure might be insufficient to resolve all compounds of interest as individual peaks. Multidimensional analysis in chromatography is a technique that combines two or more distinct separation/analysis steps, where at least one of the steps is a chromatographic separation (Marriott and Shellie 2002). Multidimensional GC (MDGC) is one of the most effective techniques used

to separate PCBs from interfering compounds, and it can be used to resolve difficulties in the separation of coeluting PCB congeners in 1DGC, as well. Moreover, it is an alternative to additional pretreatment steps that could lead to a loss of analytes (Hess et al. 1995; de Geus et al. 1996; Liem 1999b; Marriott et al. 2003). The configuration of a MDGC system consists first of a short column with a nonpolar phase to make an initial separation. The sample is chromatographed on this first column to a point just prior to the elution of unresolved peaks. Subsequently, the use of a long column with small I.D. in the second dimension can improve the separation of unresolved PCB congeners. The resolution of a MDGC is determined by the different separation power between the two phases, and increasing the differences in polarity may improve separation. As a consequence, the choice of the two columns in MDGC determines the separation power of the system (Hess et al. 1995; de Geus et al. 1996; Marriott and Shellie 2002; Marriot et al. 2003). In heart-cut MDGC, the first column flow is switched by a pressure valve into a second column packed with a more polar phase, where unresolved congeners can be subsequently separated from each other and from interfering compounds (Hess et al. 1995; de Geus et al. 1996; Haglund et al. 2008). The heart-cut process is capable of isolating a small region of primary column separation and transferring this region to a second column with higher selectivity, which permits enhancement of resolution of the heart-cut zone. This technique has been successfully used for the measurement of target analytes. However, when screening of complex samples is the main aim (i.e., when unknowns are present), heart-cut MDGC becomes extremely laborious and time consuming. An alternative is to subject the sample to a comprehensive 2DGC (GCxGC) separation (Adahchour et al. 2006). Problems associated with heart-cut MDGC analysis include the

Analysis of PCBs in Food

lack of reference data on PCB retention time for several stationary phases, retention time variability, long run times, and difficulties in using internal standard quantitation techniques. In principle, heart-cut MDGC presents a very high resolution, but the gain in total peak capacity is limited because it is only possible to perform a small number of heart-cut transfers in each run, as peaks of one cut may interfere with those of another (de Geus et al. 1996; Cochran and Frame 1999; Marriot et al. 2003; Adahchour et al. 2008); therefore, heart-cut MDGC is not a routine technique. GCxGC, also known as comprehensive MDGC or 2DGC, has evolved from heart-cut MDGC. The major benefit of GCxGC in environmental analysis is the increase of sensitivity and resolution for ultratraces of target compounds that are coextracted with impurities and interferences (Marriott and Shellie 2002; Haglund et al. 2008). A GCxGC system consists of two columns connected serially such that samples eluting from the first dimension enter in the second dimension and are analyzed sequentially. The second separation must operate at much higher speed than the first to perform hundreds of analyses during the period of the first instrument analysis. The mechanism of separation in the first dimension depends on the volatility of the analytes, whereas the second-dimension column provides class-dependent selectivity and creates an efficient separation from the matrix-related interfering compounds. Column sets are typically nonpolar (first dimension)/polar (second dimension). Because the two stationary phases differ in separation mechanisms, compounds that are not resolved on the first column may be separated on the second. Each fraction is injected onto a shorter, narrower column, with a dimension of typically 0.5– 2 m × 0.1 mm I.D. × 0.1 μm. The separation mechanism in the second column is independent from that in the first, even if stationary phases have similar polarity (Phillips and Beens 1999; Marriott et al. 2003). In particu-

35

lar, the DB-XLBxLC-50 set, provides an excellent group separation based on planarity (Adahchour et al. 2006; Haglund et al. 2008). Korytg´r et al. (2004) have studied the separation of seventeen 2,3,7,8 PCDD/PCDF and 12 DL-PCBs in milk samples, using different combinations of first- and second-dimension columns. DB-XLBxLC-50 was the best choice, allowing a good separation of the target analytes from matrix constituents. The key element of a GCxGC system is the modulator, which is positioned at the junction of the dual column set. It serves three goals: collecting fraction of peaks eluting from the first-dimension column, reinjection of the collected fractions into the seconddimension column for the separation, and finally, trapping of eluents from the first column during the launch of the preceding fraction (Kristenson et al. 2003; Marriott et al. 2003). As a result of modulator activity, the S/N ratio increases and the limit of detection (LOD) decreases compared to 1DGC (normally, S/N ratio increases 5-fold to 10fold). Temperature plays an important role in concentrating analytes into narrow bands in the modulator; elevated temperatures can accelerate this process, and lower temperatures can retard it (Marriott and Shellie 2002; Harju et al. 2003; Danielsson et al. 2005). GCxGC is more complex than heart-cut MDGC because it requires a modulator and sophisticated software (de Geus et al. 1996).

Detection The concentrations of PCBs in food are in the range of picograms per gram. Therefore, low detection limits are essential if these compounds are to be measured with accuracy and precision (Hess et al. 1995). Electron capture detector The electron capture detector (ECD) is one of the most commonly used detectors for measuring PCB concentrations after gas

36

Analysis of Endocrine Disrupting Compounds in Food

chromatographic separation due to its sensitivity and selectivity toward polyhalogenated compounds. Moreover, it is low cost and easy to operate; however, it is not recommended for non-ortho PCB analysis (Muir and Sverko 2006) because the detection limit for ECD is around 0.1 ng/g and normal levels of planar PCBs may be in the low picogram per gram range. When ECD is used for PCB analysis, it may cause nonlinear response and variation in response within a PCB congeners group. Several authors have reported the limitation of the linear range of ECD; as a consequence, a single-point calibration is not sufficient because the ultratrace concentrations of PCBs in matrices may fall in the nonlinear region of the detection. A multipoint calibration is necessary to reduce the error, particularly for lower concentrations. Variability associated with ECD response can lead to errors in quantification, and a trend toward a greater response can be observed with the increase of chlorine atoms. Another important factor that may influence the ECD response is temperature; normally an analysis with ECD is performed at 320°–340°C, but for some PCBs lower temperatures, such as 180–200°C, permit a better ECD response. The gas used must be ultrapure to protect both the GC column and the detector itself (Hess et al. 1995; Booij et al. 1998; Cochran and Frame 1999; Muir and Sverko 2006; Verenitch et al. 2007). The ability of ECD to identify a single PCB congener relies on the retention time alone; furthermore, reference 13 C12-labeled standards cannot be used (Ayris et al. 1997; van Leeuwen and de Boer 2008). The use of micro-ECD (μECD), which presents a cell size of 150 μL (one-tenth the size of ECD) reduces the effect of matrix contamination on the detector response; in fact, conventional ECD cell volume can cause severe peak broadening (Cochran and Frame 1999; Korytg´r et al. 2006). To avoid band broadening it is necessary to use very high detector temperature (300°C)

and high flow of make-up gas (150 mL/min) (Kristenson et al. 2003). GCxGC/μECD analysis can provide a full PCB congener profile. This congener pattern faithfully reproduces the profiles obtained by GC-HRMS, and this technique could also be used as a routine method for PCB analysis in food. Unfortunately, the current EC legislation recognizes only HRGC/HRMS as confirmatory methods for official control of dioxins and PCBs, and at present, this technique is 10 times more sensitive. Mass spectrometry Today, GC-HRMS is considered a point of reference for the accurate and specific analysis of PCBs in environmental and food samples because of the required selectivity and detection limits (picogram per gram level). Nevertheless, GC-HRMS instrumentation is very expensive, and qualified personnel are needed (Malavia et al. 2004; Gómara et al. 2006). The benefit of mass spectrometry (MS) techniques is the improved identification of PCBs as compared to the ECD method. In the MS technique, the compounds are ionized with the production of characteristic molecular or fragment ions, which are separated on the basis of their mass-to-charge ratio (m/z). The ionization and fragmentation of the molecules can be obtained by using two techniques: electronic ionization (EI) and electron capture negative ionization (ECNI). The EI of PCBs produces molecular ion isotope patterns, which are used for qualitative and quantitative purposes, and fragments, which depend on the PCBs’ structure. Büthe and Denker (1994) have studied the stability of single PCBs versus fragmentation when EI is applied: non-, mono-, and tetra-ortho substitutions stabilize the molecules more than do di- or tri-ortho substitution. Di-ortho substitution on one ring of the system makes the PCBs more stable than di-ortho substitution proportionally distributed on both rings. Para-Cl substitution stabilizes PCBs in

Analysis of PCBs in Food

general, and symmetrical distribution of Cl atoms increases the stability. Negative ions of PCBs(ECNI) are produced by capture of low-energy electrons generated in the MS source from a buffer gas such as methane. The gas acts to stabilize negative ions, and they do not dissociate prior to their mass analysis. When ECNI is used, sensitivity is enhanced, and the technique is selective because polyhalogenated compounds preferentially form negative ions. The analysis can be made with high-resolution or low-resolution instruments (van Leeuwen and de Boer 2008). With GC low-resolution mass spectrometry (LRMS), PCBs can be measured at low picogram concentrations using EI in selected ion mode (SIM). In fact, residue analysis requires the ability to detect small amounts of substances, and in this case the SIM mode is widely recognized as the method of choice (Böthe and Denker 1994; Muir and Sverko 2006). GC-LRMS is a screening method used to measure PCBs in foodstuff, and HRGC/HRMS is the confirmatory method required by EC. In particular, for DL-PCBs, LODs should be in the low picogram range, and the compounds should be resolved from other interfering compounds, the concentrations of which may be up to several orders of magnitude higher than those of target analytes (EU Commission Regulation 1883/2006/EC). Ion trap mass spectrometry and ion trap tandem mass spectrometry The ion trap mass spectrometry (ITMS) analyzer is a device in which the ions are stored in an electric field and then destabilized/ ejected, according to their mass, to a detector. Full-scan ITMS data can be acquired at very low analyte levels (picograms). In ITMS the presence of interfering compounds that are coeluted with PCBs, even if they don’t have the same mass as target molecules, can reduce the sensitivity (Cochran and Frame 1999; Liem 1999b). Ion trap can operate as a tandem

37

mass spectrometry (MS/MS) analyzer to improve the selectivity of the system (Ahmed 2003). The use of MS/MS has never gained widespread popularity because of its rather expensive and complicated nature, but the introduction of ion trap MS/MS detection has removed these obstacles (Leonards et al. 1996). According to Gómara et al. (2006), gas chromatography-ion trap tandem mass spectrometry (GC-ITD-MS/MS) has proved to be superior to GCxGC/μECD in the determination of DL-PCBs in complex matrices, mainly because of the higher selectivity associated with the ITD-MS/MS process. ITD-MS/MS should prevent the loss of sensitivity because all ion’s products remain in the trap during the complete ionization process. Satisfactory results were also obtained for the less abundant non-ortho PCBs. Thanks to selectivity, sensitivity, and stability, this recent technique can be used for routine analysis of PCBs in food. Verenitch et al. (2007) have analyzed fish samples, and the results obtained in their study demonstrate that GC-ITD-MS/MS provides higher data quality than those achievable by GC-ECD. For this particular set of target analytes, the specificity achievable with GC-ITD-MS/MS was comparable to that obtained by GC-HRMS, and both techniques provided comparable data in terms of accuracy and precision. Malavia et al. (2004) have evaluated and demonstrated the suitability of GC-ITD-MS/ MS for the analysis of non-ortho PCBs in fish samples. Moreover, the comparison of results obtained by GC-ITD-MS/MS and GC-HRMS showed that GC-ITD-MS/MS is an interesting lower cost alternative to GC-HRMS for the analysis of non-ortho PCBs. Time-of-flight mass spectrometry Other alternative methods to GC-HRMS exist, and one of most promising is timeof-flight mass spectrometry (TOF-MS). As

38

Analysis of Endocrine Disrupting Compounds in Food

compared to SIM with sector instruments, TOF-MS offers more MS information because it monitors all masses at once within a range. The coupling of GCxGC separation and TOF-MS detection presents no difficulties, and there is no peak broadening. Consequently, the GCxGC-TOF-MS coupling is a powerful instrument combining improved chromatographic resolution of GCxGC and the analytical resolving power of the TOF-MS. Unfortunately, TOF-MS instruments are very expensive (Focant et al. 2004b; Korytg´ r et al. 2006). Focant et al. (2004c) have studied the separation of 209 PCB congeners using GCxGC-TOF-MS. Four column combinations have been used, and the HT-8/BPX-50 set produced the best separation. A total of 192 congeners were resolved in 146 min using this column set, and the 12 most toxic PCBs and the 7 marker PCBs were separated from any interfering congeners. Focant et al. (2005) have measured with GCxGC-IDTOF-MS the concentration of four non-ortho and eight mono-ortho PCBs and six indicator PCBs in foodstuff samples. GCxGC-IDTOF-MS showed its suitability to offer quality control capability.

Other methods of analysis EU Commission Regulation 1883/2006/EC allows the use of screening methods to select “samples with significant levels of dioxin and DL-PCBs.” These samples then are analyzed with a confirmatory method (HRGC/HRMS) to quantify dioxin and DL-PCB concentration and checked to see if the sample is below or above the maximum tolerable limits as defined by Commission Regulation (EC) No. 1881/2006. The procedures using chemical analysis are time consuming and very expensive, require a cleanup step to remove interfering compounds, and require the use of sophisticated expensive instruments, such as GCHRMS. For these reasons, the use of other

detection methods may be considered to study PCBs’ presence in foods. Immunoassays Immunoassays (IAs) are based on highly specific binding of antibodies to some analytes. Antibodies are produced by the immune system after exposure of animals to foreign substances; by immunizing animals with a hapten, it is possible to produce antibodies that can be used as specific reagents to recognize the analyte. The detection can be made using a radioactive tracer (radioimmunoassay, or RIA) or by the reaction of an enzyme linked to antibody or antigen (enzyme immunoassay, or EIA). These methods feature an easy extraction, are rapid, and do not use cleanup procedures. In comparison to cellbased bioassays, the most important advantages are speed, simplicity, low cost and parallel processing of many samples, easy automatization, and potential field use. Disadvantages are reported as being costly development, presence of cross-reacting compounds, and nonspecific interferences. All EIAs are characterized by lack of absolute specificity, and the test designer must determine a calibration procedure for every matrix to take into account cross-reactivity patterns (Díaz-Ferrero et al. 1997; Behnisch et al. 2001). Many methods are described as being semiquantitative for screening clean samples. In particular, an ELISA (enzyme-linked immunosorbent assay) uses an enzyme, horseradish peroxidase (HRP), as the PCB conjugate. These techniques are extremely sensitive, inexpensive, and easy to perform. ELISAs are based on competitive binding in which the binder molecule, an excess amount of labeled analyte or coated antigen, and target analyte are allowed to approach equilibrium. The sample antigen competes with the coated antigen for binding sites on the labeled antibody. After the wash step, the detection is performed by adding a chromophore, and quantification is performed via

Analysis of PCBs in Food

spectrophotometric measurements (Muir and Sverko 2006). Zajicek et al. (2000) have compared ELISA to GC for measurement of PCBs in fish extract. They concluded that results suggested that higher chlorinated PCB congeners have high affinity for the anti-PCB antibodies and that the ELISA-derived PCB concentrations are dependent on the degree of chlorination. Fillmann et al. (2002) have compared the results from PCB analyses of mussel tissue extracts by ELISA and conventional GC-ECD. They have described a strong correlation between total PCBs quantified with both techniques. Differences between techniques did occur when GC results were corrected for procedural recovery (based on recoveries of internal standards). Galloway et al. (2002) have described, after analysis of mussel tissue extracts by ELISA, a strong correlation between results from the PCB immunoassay and GC-ECD and concluded that the immunoassay has sufficient sensitivity and accuracy across a wide range of environmental concentrations. Frg´ nek et al. (2001) have studied the direct competitive ELISA, which uses antibodies with higher specificity to the most toxic coplanar PCB congeners and non-ortho PCBs with 3, 4, 3′and 3, 4, 4′-chlorination. The highly specific analyte–antibody interaction can dramatically simplify the sample preparation methods by eliminating expensive and time-consuming purification steps, but additional experiments are needed in order to verify a reliable relationship between ELISA response and the coplanar congener concentrations. Bioassays Bioassays are based on the mechanism of toxicity. Bioassays present the following advantages: rapid determination of the total potency of AhR agonist; short procedure time; low cost; high sensitivity, often at picogram level; and ability to predict the outcome of in vivo studies in terms of magnitude of

39

effect but not the total spectrum of action. The disadvantages include limited validation data, questions about the degree of reliability, limited cross-laboratory validation studies with the same technology, lack of crossvalidation studies with different bioassays, and lack of internationally evaluated quality criteria. However, in vitro tests, such as CALUX and EROD, offer a more rapid, sensitive, and relatively inexpensive solution than does an in vivo test system (Behnisch et al. 2001). At present, CALUX is the best screening method for DL-PCBs and dioxins in food and the only assay used in routine monitoring and during incidents. This assay uses genetically modified rat or mouse hepatoma cells that respond to compounds that can bind AhR; receptor binding causes the activation of transcription factors and thus development of toxicity and gene expression. The recombinant CALUX cells contain the transfected AhR-responsive firefly luciferase reporter gene, which responds to dioxin and DL-PCBs. However the CALUX assay can respond to many AhR agonists, and to improve the selectivity of the test, a cleanup procedure for extraction and purification of the sample is necessary (Hoogenboom et al. 2006). Scippo et al. (2004) have studied the use of DR-CALUX (dioxin-responsive CALUX) to screen samples for dioxins, furans, and DL-PCBs using a quantitative approach, and the results obtained were compared with those of GC-HRMS. Comparison of results of cell bioassay and GC-HRMS are difficult because these techniques are based on different principles; cell-based bioassay evaluates the biological effects in vitro of a complex mixture extracted and purified from a sample, whereas GC-HRMS detects the quantity of substances that are present in the mixture. Novel techniques, such as TOF-MS, in combination with CALUX, may contribute to speeding up the identification of compounds that produce a response. The CALUX assay is suitable for high-throughput screening and

40

Analysis of Endocrine Disrupting Compounds in Food

detection of samples with levels higher than common background (Hoogenboom et al. 2006). The EROD method measures the binding of DL compound to AhR and the subsequent induction of CYP1A by related deethylation of 7-ethoxyresorufin to resorufin. The analysis by this method can be made in vitro or in vivo. In vivo bioassays have the objective of measuring the effect in animals exposed to contaminants, whereas in vitro analyses are carried out in culture cells (Díaz-Ferrero et al. 1997). Several cell lines are used for EROD bioassays, such as the rat cell line, chicken embryo hepatocytes, cultured chicken embryo hepatocytes, fish cell lines, and human hepatoma cells (Behnisch et al. 2001). For the purpose of risk-assessment of DL-PCBs, Tsang et al. (2009) have studied, using a micro-EROD assay, DL-PCBs in nine groups of food items (freshwater fish, marine fish, pork, chicken, eggs, leafy and nonleafy vegetables, rice, and flour) and in three types of human samples. They concluded that the micro-EROD assay provides a rapid, costeffective preliminary assessment of dioxin and DL-PCB levels in both food and human samples. IAs and the CALUX bioassay are cheaper, easier, and more rapid than conventional instrumental methods. Despite the advantages, there are some points that should be further studied: cross-reactivity for IAs, influence of organic solvent in the assays, need for a cleanup step, and differences between cell lines from different species.

Concentration of PCBs in food Distribution of PCBs PCBs occur in the environment as a mixture of different congeners. Current releases in the environment are mainly as a result of the recycling of these persistent contaminants from soil to air. Following emission in the atmosphere, PCBs can be subjected to long-

range atmospheric transport before redeposition, away from the area of their production and use. However, there are differences in the characteristic travel distances of different congeners (Van Pul et al. 1998; Beyer et al. 2000), and in general, lighter congeners can undergo greater long-range transport because they are deposited less by dry gaseous and particulate deposition. As a consequence, lighter congeners have become better mixed in the atmosphere than heavier congeners (Beyer et al. 2000). Due to their lipophilic properties, these compounds can accumulate in the food chain, in particular in lipid-rich foods of animal origin, such as meat, dairy products, and fish, where they can reach high concentrations. On the contrary, food items with high water content, such as fruit and vegetables, have generally lower PCB concentrations. Diet is by far the main source of exposure of the population to PCBs, and total intakes may vary greatly in relation to the kind of diet eaten by a particular population. Because fish and fish products are particularly rich in PCBs, these food items are generally important contributors to the total dietary exposure. In particular, DL-PCBs are widely distributed contaminants of the aquatic environments and can be accumulated in aquatic organisms through the food chain. Marine organisms are exposed to these contaminants by contact with seawater and sediments or by ingestion of contaminated prey (Moon and Ok 2006), and consumers can eventually be exposed to PCBs by consumption of contaminated seafood. Generally, PCB concentrations in fish and other foods of animal origin are higher than in fruit and vegetables, but because some diets, such as the Mediterranean diet, are rich in fruit and green vegetables, these too might contribute significantly to the total dietary intake of these substances (Zuccato et al. 1999). However, where marine products are important components of the diet, for instance in Nordic European countries such as Sweden

Analysis of PCBs in Food

and Finland, and in Japan, fish and fish products account for up to 70–80% of total PCB intake (Kirivanta et al. 2004; Darnerud et al. 2006). The contrary has been observed in other countries such as Poland, where fish consumption is lower, and the contribution of this food item to the total PCB intake seems to be less significant (Usydus et al. 2009). Meat and dairy products are other important contributors to the dietary intake, and butter has been frequently used to assess the exposure of the population to PCBs through these items. For instance, Kalantzi et al. (2001) have measured PCB levels in butter worldwide and have shown that ΣPCB concentrations were lowest in samples from Australia/New Zealand and highest in samples from the Czech Republic by a factor of 60 (difference between the lowest and the highest). Therefore, this study identified particularly high levels of PCBs in eastern Europe, where the production and use of these molecules stopped later than in the rest of Europe. Moreover, high levels in some countries might represent losses from aging or obsolete electrical materials or from hazardous waste repositories. In general, butter samples from Europe had higher concentrations than those from the United States, and samples from the Southern Hemisphere consistently contained lower concentrations than those from the Northern Hemisphere, reflecting the greatest historical production and use of PCBs in this area of the globe. Moreover, European samples contained higher proportions of highly chlorinated congeners (CBs 180, 153, and 138). In contrast, samples from Australia, the Philippines, Canada, and Brazil had higher proportions of lighter congeners (tetra- and pentachlorinated). Within-country variability in PCB concentrations was also quite high, particularly for larger countries, reflecting differences in urbanization and industrialization within geographical regions. For example, butter from the U.S. East Coast contained higher PCB concentrations than samples from the West Coast or the Midwest,

41

probably reflecting differences in industrial activity and population density. Fruit and vegetables may grow in soil contaminated by the atmospheric deposition of volatile and semivolatile organic compounds, including PCBs. Levels in vegetables might therefore reflect local contamination. However, Zohair et al. (2006) have measured PCBs in different carrot and potato species, observing a positive correlation between concentrations in soil and in carrots but not in potatoes. This observation suggests that some species’ characteristics were important in determining the uptake from the soil. Moreover, according to Chiou et al. (2001), the lipid content of plants is the most important determinant of the uptake of lipophilic compounds.

Toxicity of PCBs Within the PCB family, degree and patterns of chlorine substitution lead to different types of toxicity. Twelve PCBs, the so-called DLPCBs (four non-ortho and eight mono-ortho substitutes, including CBs 77, 81, 126, 169 and CBs 105, 114, 118, 123, 156, 157, 167, 180), have chemical structure and toxicity similar to that of dioxins. Number and position of chlorine atoms in these compounds can be related to their toxicity aspects; a chlorine substitution pattern allows DL-PCBs to adopt a planar geometry that permits them to bind to the aryl hydrocarbon receptor (AhR). This receptor is an important regulator of physiological and developmental processes (Fernandez-Salguero et al. 1997). The AhR takes part in pivotal upstream events of the apoptosis cascade (Nebert et al. 2000; Dong et al. 2004), exerts an important level of influence on reproductive success (Abbott et al. 1999), and participates in cell cycle regulation (Marlowe et al. 2004). Inappropriate activation of AhR by exogenous ligands, such as PCBs, has been shown to cause disruption of many cellular and developmental processes, including cell proliferation and differentiation (Barouki et al.

42

Analysis of Endocrine Disrupting Compounds in Food

2007). The capacity of AhR agonist to cause endocrine disruption has been demonstrated in both the male and female reproductive systems (Hotchkiss et al. 2008); hormone signaling pathways may be sensitive to extremely low-dose exposure, and DL compounds are strongly correlated with disruption of estrogen-mediated signaling (Safe et al. 1998). In the mammalian fetus, the AhR plays an important role in both resolving vascular structures and mediating cardiovascular toxicity of dioxin-like compounds (Bello et al. 2004). Cardiotoxicity can be the consequence of high exposure to these molecules during the prenatal period; however, some epidemiological studies have suggested a link between DL compounds and cardiotoxicity following occupational exposure (Reichard et al. 2005). Although DL compounds are not genotoxic in the Ames test, some studies suggest that the pathway of their toxic effects involves oxidative DNA damage and increased mutation frequency (Cairns et al. 1991); therefore, DL-PCBs have toxicity comparable to that of dioxins and are included in the toxic equivalency (TEQ) concept that is the basis for

health risk assessment of DL compounds. This is a system for ranking the relative potencies of the DL activity of DL-PCBs. For the remaining PCB congeners, referred to as nondioxin-like PCBs (NDL-PCBs), which are the most abundant PCBs in the environment and human tissues, no such tool has yet been developed because their toxicological profile has not been completely clarified. These congeners have been reported to induce reduction of dopamine neurotransmitter levels and to interfere with calcium homeostasis in neuronal cells (Shain et al. 1991), suggesting that they might have a different mode of action with some direct effects on neurons (Brown et al. 1998; Seegal 1998; Tilson et al. 1998). Threshold limits for the sum of dioxin and DL-PCBs in foodstuffs have been set by EU Commission Regulation (EC) 1881/2006 of 19 December 2006 (Table 2.2). At the moment, no limits have been set for NDLPCBs. However, the EU Commission is considering setting maximum levels for NDL-PCBs expressed as the sum of six indicator congeners (PCB 28, 52, 101, 138, 153, and 180).

Table 2.2. Threshold limits for the sum of dioxin and DL-PCBs in foodstuffs in EU. Foodstuffs Fat of the following animals: • bovine animals and sheep • poultry • pigs Mixed animals fat Vegetables oils and fats Marine oils intended for human consumption Fish liver and derived products thereof, with the exception of marine oils Meat and meat products (excluding edible offal) of the following animals: • bovine animals and sheep • poultry • pigs Liver of terrestrial animals and derived products thereof Muscle meat of fish and fishery products and products thereof, excluding eel Muscle meat of eel and products thereof Raw milk and dairy products, including butterfat Hen eggs and egg products

Sum of dioxins and dioxin-like PCBs (WHOPCDD/F-PCB-TEQ) 4.5 pg/g fat 4.0 pg/g fat 1.5 pg/g fat 3.0 pg/g fat 1.5 pg/g fat 10.0 pg/g fat 25.0 pg/g wet weight (upper bound) 4.5 pg/g fat 4.0 pg/g fat 1.5 pg/g fat 12.0 pg/g fat 8.0 pg/g wet weight 12.0 pg/g wet weight 6.0 pg/g fat 6.0 pg/g fat

WHOPCDD/F-PCB-TEQ, World Health Organization PCDD/F-PCB toxic equivalents.

Analysis of PCBs in Food

References Abbott BD, Schmid JE, Pitt JA, Buckalew AR, Wood CR, Held GA, Diliberto JJ. Adverse reproductive outcomes in the transgenic AhR deficient mouse. Toxicology and Applied Pharmacology 1999, 155:62–70. Abrha Y, Raghavan D. PCB recovery from spiked organic matrix using accelerated solvent extraction and Soxhlet extraction. Journal of Hazardous Materials 2000, B80:147–157. Adahchour M, Beens J, Brinkman UATh. Recent developments in the application of comprehensive two dimensional gas chromatography. Journal of Chromatography A 2008, 1186:67–108. Adahchour M, Beens J, Vreuls RJJ, Brinkman UATh. Recent developments in comprehensive twodimensional GC: Introduction and instrumental set-up. Trends in Analytical Chemistry 2006, 25: 438–454. Agustin MRR, Park HM, Hong SM, Ryu JC, Lee KB. Determination of mono-to octachlorobiphenyls in fish oil using Florisil adsorption followed by headspace solid-phase microextraction and gas chromatography with TOF spectrometric detection. Journal of Chromatography A 2005, 1085:278–284. Ahmed FE. Analysis of pesticides and their metabolites in foods and drinks. Trends in Analytical Chemistry 2001, 20:649–661. Ahmed FE. Analysis of polychlorinated biphenyls in food products. Trends in Analytical Chemistry 2003, 22:170–185. Alcock RE, Halsall CJ, Harris CA, Johnston AE, Lead WA, Sanders G, Jones KC. Contamination of environmental sample prepared for PCB analysis. Environmental Science and Technology 1994, 28: 1838–1842. Antunes P, Gil O, Bernardo-Gil MG. Supercritical fluid extraction of organochlorines from fish muscle with different sample preparation. Journal of Supercritical Fluids 2003, 25:135–142. Asmund G, Vorkamp K, Backus S, Comba M. An update on analytical methods, quality assurance and quality control used in the Greenland AMAP programme: 1999–2002. Science of the Total Environment 2004, 331:233–245. Ayris S, Currado GM, Smith D, Harrad S. GC/MS procedures for the determination of PCBs in environmental matrices. Chemosphere 1997, 35:905–917. Barker SA. Application of matrix solid-phase dispersion in food analysis. Journal of Chromatography A 2000a, 880:63–68. Barker SA. Matrix solid-phase dispersion. Journal of Chromatography A 2000b, 885:115–127. Barouki R, Coumoul X, Fernandez-Salguero PM. The aryl hydrocarbon receptor, more than a xenobioticinteracting protein. FEBS Letters 2007, 581: 3608–3615. Battu RS, Balwinder S, Kang BK. Contamination of liquid milk and butter with pesticides residues in Ludhiana district of Punjab state, India. Ecotoxicology and Environmental Safety 2004, 59:324–331.

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Bayarri S, Turrio Baldassarri L, Iacovella N, Ferrara F, Di Domenico A. PCDDs, PCDFs, PCBs, DDE in edible marine species from the Adriatic Sea. Chemosphere 2001, 43:601–610. Behnisch PA, Hosoe K, Sakai SI. Bioanalytical screening methods for dioxins and dioxin-like compounds: A review of bioassay/biomarker technology. Environmental International 2001, 27:413–439. Bello SM, Heiderman W, Peterson RE. 2,3,7,8 TCDD inhibits regression of the common cardinal vein in developing zebrafish. Toxicological Sciences 2004, 78:258–266. Beyer A, MacRay D, Matthies M, Wania F, Webster E. Assessing long-range transport potential of persisting organic pollutant. Environmental Science and Technology 2000, 34:699–703. Beyer A, Biziuk M. Application of sample preparation techniques in the analysis of pesticides and PCBs in food. Food Chemistry 2008, 108:669–680. Bianco G, Novario G, Bochicchio D, Anzilotta G, Palma A, Cataldi TRI. PCBs in contaminated soil samples evacuate by GC-ECD with dual column and GCHRMS. Chemosphere 2008, 73:104–112. Björklund E, Müller A, von Holst C. Comparison of fat retainers in accelerated solvent extraction for the selective extraction of PCBs from fat-containing samples. Analytical Chemistry 2001, 73:4050–4053. Björklund E, Sporring S, Wiberg K, Haglund P, von Holst C. New strategies for extraction and clean up of persistent organic pollutant from food and feed samples using selective pressurized liquid extraction. Trends in Analytical Chemistry 2006, 25:318–325. Björklund E, von Holst C, Anklam E. Fast extraction, clean up and detection methods for the rapid analysis and screening of seven indicator PCBs in food matrices. Trends in Analytical Chemistry 2002, 21:39–52. Bogialli S, Di Corcia A. Matrix solid-phase dispersion as valuable tool for extracting contaminants from foodstuffs. Journal of Biochemical and Biophysical Methods 2007, 70:163–179. Booij K, Hillebrand MTJ, van Weerlee EM. Calibration of the electron-capture detector for the determination of polychlorinated biphenyls. Analyst 1998, 123:145–420. Borgå K, Gabrielsen GW, Skaare JU. Biomagnification of organochlorines along Barents Sea food chain. Environmental Pollution 2001, 113:187–198. Brown AP, Oliviero-Verbel J, Holdan WL, Ganey PE. Neutrophil activation by polychlorinated biphenyls: Structure-activity relationship. Toxicological Sciences 1998, 46:308–316. Bucholski KA, Begerow J, Winneke G, Dunemann L. Determination of polychlorinated biphenyls and chlorinated pesticides in human body fluids and tissues. Journal of Chromatography A 1996, 754:479–485. Buldini PL, Ricci L, Lal Sharma J. Recent applications of sample preparation techniques in food analysis. Journal of Chromatography A 2002, 975:47–70. Büthe A, Denker E. Qualitative and quantitative determination of PCB congeners by using a HT-5 GC column and an efficient quadrupole MS. Chemosphere 1994, 30:753–771.

44

Analysis of Endocrine Disrupting Compounds in Food

Cairns W, Cairns C, Pongratz I, Poellinger L, Okret S. Assembly of glucocorticoid receptor complex prior to DNA binding enhances its specific interaction with glucocorticoid response element. Journal of Biological Chemistry 1991, 226:11221–11226. Camel V. Microwave-assisted solvent extraction of environmental samples. Trends in Analytical Chemistry 2000, 19:229–247. Camel V. Recent extraction techniques for solid matricessupercritical fluid extraction, pressurized fluid extraction, and microwave-assisted extraction: Their potential and pitfalls. Analyst 2001, 126:1182–1193. Carabias-Martínez R, Rodríguez-Gonzalo E, RevillaRuiz P, Hernández-Méndez J. Pressurized liquid extraction in the analysis of food and biological samples. Journal of Chromatography A 2005, 1089: 1–17. Carro N, Garcia I, Ignacio MC, Llompart M, Yebra MC, Mouteira A. Microwave-assisted extraction and mild saponification for determination of organochlorine pesticides in oyster samples. Analytical and Bioanalytical Chemistry 2002, 374:547–553. Carro N, Garcìa I, Llompart M. Closed-vessel assisted microwave extraction of polychlorinated biphenyls in marine mussels. Analusis 2000, 28:720–724. Castello G, Testini G. Determination of retention indices of polychlorobiphenyls by using other compounds detectable by electron-capture detection or selected polychlorobiphenyls as the reference series. Journal of Chromatography A 1996, 741:241–249. Cencicˇ Kodba Z, Brodnjak Voncˇina D. A rapid method for the determination of organochlorine, pyrethroid pesticides and polychlorobiphenyls in fatty foods using GC with electron capture detection. Chromatographia 2007, 66:619–624. Centi S, Silva E, Laschi S, Palchetti I, Mascini M. Polychlorinated biphenyls detection in milk samples by an electrochemical magneto immunosensor coupled to solid-phase extraction and dispensable low density array. Analytica Chimica Acta 2007, 594:9–16. Chiou CT, Sheng Gy, Manes M. A partition-limited model for the plant uptake of organic contaminants from soil and water. Environmental Science and Technology 2001, 35:1437–1444. Cochran JW, Frame G. Recent developments in the high resolution GC of PCBs. Journal of Chromatography A 1999, 843:323–368. Corsolini S, Romeo T, Ademollo N, Greco S, Focardia S. POPs in key species of marine Antarctic ecosystem. Microchemical Journal 2002, 73:187–193. Covaci A, Schepens P. Simplified method for determination of organochlorine pollutants in human serum by solid-phase disk extraction and gas-chromatography. Chemosphere 2001, 439–447. Crouch MD, Barker SA. Analysis of toxic wastes in tissue from aquatic species application of matrix solidphase dispersion. Journal of Chromatography A 1997, 774:287–309. Danielsson C, Wiberg K, Korytár P, Bergek S, Brinkman UAT, Haglund P. Trace analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans, and WHO poly-

chlorinated biphenyls in food using comprehensive two-dimensional gas chromatography with electroncapture detection. Journal of Chromatography A 2005, 1085:61–70. Darnerud PO, Atuma S, Aune M, Bjerselius R, Glynn A, Petersson Gravè K, Becker W. Dietary intake estimations of organohalogen contaminants based on Swedish market basket data. Food and Chemical Toxicology 2006, 44:1597–1606. Dean JR, Xiong G. Extraction of organic pollutants from environmental matrices: Selection of extraction technique. Trends in Analytical Chemistry 2000, 19: 553–564. de Boer J, Law RJ. Developments in the use of chromatographic techniques in marine laboratories for the determination of halogenated contaminants and polycyclic aromatic hydrocarbons. Journal of Chromatography A 2003, 1000:223–251. de Boer J. Capillary gas chromatography for the determination of halogenated micro-contaminants. Journal of Chromatography A 1999, 843:179–198. de Boer J. Chlorobiphenyls in bound and non-bound lipids of fishes; comparison of different extraction methods. Chemosphere 1988, 17:1803–1810. De Geus HJ, de Boer J, Brinkman UATh. Multidimensionality in gas chromatography. Trends in Analytical Chemistry 1996, 15:168–178. Díaz-Ferrero J, Rodriguez-Larena MC, Comellas L, Jimenez B. Bioanalytical methods applied to endocrine disrupting PCBs, PCDD and PCDF: A review. Trends in Analytical Chemistry 1997, 16: 563–573. Dong Y, Sun C, Xu S, Dai J, Fen J, Jiang X, Wang L. Polychlorinated organic compounds (PCOCs) in the Yangtse River water samples using SPE and GC/ECD. Bulletin of Environmental Contamination and Toxicology 2000, 64:383–389. Dong W, Teraoka H, Tsujimoto Y, Stageman JJ, Hiraga T. Role of AhR in mesencephalic circulation failure and apoptosis in zebrafish embryos exposed to 2,3,7,8 TCDD. Toxicological Sciences 2004, 77:109–116. Dorne JLCM, Bordajandi LR, Amzal B, Ferrari P, Verger P. Combining analytical techniques, exposure assessment, and biological effects for risk assessment of chemicals in food. Trends in Analytical Chemistry 2009, 28:695–707. Environmental Protection Agency (EPA). Method 1668 Revision B, November, 2008. Chlorinated PCB congeners in water, soil sediment, biosolids, and tissue by HRGC-HRMS. Available at: www.epa.gov/ waterscience/methods/method/files/1668.pdf European Union (EU). Commission Regulation No. 1883/2006 of 19 December 2006. Laying down methods of sampling and analysis for the official control of the levels of dioxins and dioxin-like PCBs in certain foodstuffs. Official Journal of the European Union L 364:32–43. European Union (EU). Commission Regulation No. 1881/2006 of 19 December 2006. Setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union L 364:5–24. Fernandes A, White S, D’Silva K, Rose M. Simultaneous

Analysis of PCBs in Food

determination of PCDDs, PCDFs, PCBs, and PBDEs in food. Talanta 2004, 63:1147–1155. Fernandez-Salguero PM, Ward JM, Sundberg JP, Gonzales FJ. Lesion of AhR-deficent mice. Veterinary Pathology 1997, 34:605–614. Fildalgo-Used N, Blanco-González E, Sanz-Medel A. Sample handling strategies for the determination of persistent trace organic contaminants from biota samples. Analytica Chimica Acta 2007, 590:1–16. Fillmann G, Galloway T, Sanger RC, Depledge MH, Readman JW. Relative performance of immunochemical (ELISA) and GC-ECD techniques to quantify PCBs in mussel tissues. Analytica Chimica Acta 2002, 461:75–84. Focant JF, Eppe G, Pirard C, De Pauw E. Fast clean up for polychlorinated dibenzo-p-dioxins, dibenzofurans, and coplanar polychlorinated biphenyls analysis of high fat content biological samples. Journal of Chromatography A 2001, 925:207–221. Focant JF, Eppe G, Pirard C, Massart AC, André JE, De Pauw E. Level and congeners distribution of PCDDs, PCDFs and non-ortho PCBs in Belgian foodstuffs. Assessment of dietary intake. Chemosphere 2002, 48:167–179. Focant JF, Eppe G, Scippo ML, Massart AC, Pirard C, Maghuin-Rogister G, De Pauw E. Comprehensive two dimensional gas chromatography with isotope dilution time-of-flight mass spectrometry for the measurement of dioxins and polychlorinated biphenyls in foodstuff comparison with other methods. Journal of Chromatography A 2005, 1086:45–60. Focant JF, Pirard C, De Pauw E. Automated sample preparation-fractionation for the measurement of dioxin and related compounds in biological matrices: A review. Talanta 2004a, 63:1101–1113. Focant JF, Reiner EJ, MacPherson K, Kolic T, Sjödin A, Patterson Jr DG, Reese SL, Dorman FL, Cochran J. Measurement of PCDDs, PCDFs, and non-orthoPCBs by comprehensive two dimensional gas chromatography-isotope dilution time-of-flight mass spectrometry (GCxGC-IDTOFMS). Talanta 2004b, 63:1231–1240. Focant JF, Sjödin A, Patterson Jr DG. Improved separation of 209 polychlorinated biphenyls congeners using GCxGC-TOFMS. Journal of Chromatography A 2004c, 1040:227–238. Folch I, Vaquero MT, Comellas L, Broto-Puig F. Extraction and clean up methods for improvement of the chromatographic determination of polychlorinated biphenyls in sewage sludge amended soils: Elimination of lipids and sulphur. Journal of Chromatography A 1996, 719:121–130. Fránek M, Deng A, Kolá V, Socha J. Direct competitive immunoassays for the coplanar PCBs. Analytica Chimica Acta 2001, 444:131–142. Galloway T, Sanger RC, Smith KL, Fillmann G, Readman JW, Ford TE, Depledge MH. Rapid assessment of marine pollution using multiple biomarkers and chemical immunoassays. Environmental Science Technology 2002, 36:2219–2226. Gilbert-López B, García-Reyes J, Molina-Diaz A. Sample treatment and determination of pesticides resi-

45

dues in fatty vegetable matrix: A review. Talanta 2009, 79:109–128. Gómara B, Fernández MA, González MJ, Ramos L. Feasibility of gas chromatography-ion trap tandem mass spectrometry for the determination of polychlorinated biphenyls in food. Journal of Separation Science 2006, 29:123–130. Gómez-Ariza JL, Bujalance M, Giráldez I, Velasco A, Morales E. Determination of polychlorinated biphenyls in biota samples using simultaneous pressurized liquid extraction and purification. Journal of Chromatography A 2002, 946:209–219. Goñi F, López R, Etxeandia A, Millán E, Vives A, Amian P. Method for the determination of selected organochlorine pesticides and polychlorinated biphenyls in human serum based on a gel permeation chromatographic clean up. Chemosphere 2009, 76:1533–1539. Grassi P, Fattore E, Generoso C, Carubelli G, Fanelli R. Rosemary plant as a bio-accumulator for monitoring of PCB contamination. Organohalogen Compounds 2008, 70:1969–1972. Haglund P, Korytár P, Danielsson C, Diaz J, Wiberg K, Leonards P, Brinkman UATh, de Boer J. GCxGCECD: A promising method for the determination of dioxins and dioxin-like PCBs in food and feed. Analytical and Bioanalytical Chemistry 2008, 390:1815–1827. Hale RC, Gaylor MO. Determination of PCBs in fish tissue using supercritical fluid extraction. Environmental Science and Technology 1995, 29: 1043–1047. Harju M, Danielsson C, Haglund P. Comprehensive two dimensional gas chromatography of the 209 polychlorinated biphenyls. Journal of Chromatography A 2003, 1019:11–126. Hennion MC. Solid-phase extraction: Method development, sorbent, and coupling with liquid chromatography. Journal of Chromatography A 1999, 856:3–54. Hess P, de Boer J, Cofino WP, Leonards PEG, Wells DE. Critical review of the analysis of non-ortho and monoortho chlorobiphenyls. Journal of Chromatography A 1995, 703:417–465. Hoogenboom L, Goeyens L, Carbonelle S, van Loco J, Beernaert H, Baeyens W, Traag W, Bovee T, Jacobs G, Schoeters G. The CALUX bioassay: Current status of its application to screening food and feed. Trends in Analytical Chemistry 2006, 25:410–420. Hotchkiss AK, Rider CV, Blystone CR, Wilson VS, Harting PC, Ankley GT, Foster PM, Gray LE. Fifteen years after Wingspread–Environmental endocrine disrupters and human and wildlife health: Where we are today and where we need to go. Toxicological Sciences 2008, 105:235–259. Huckins J, Tuberger M, Lebo J, Gale R, Schwartz T. Semipermeable membrane devices containing model lipid: A new approach to monitoring the bioavailability of lipophilic contaminants and estimating their bioconcentration potential. Chemosphere 1990, 20: 533–552. Hummert K, Vetter W, Luckas B. Fast and effective sample preparation for determination of organochlorine compounds in fatty tissue of marine mammals

46

Analysis of Endocrine Disrupting Compounds in Food

using microwave extraction. Chromatographia 1996, 42:300–304. Isosaari P, Hallikainen A, Kirivanta H, Vuorinen PJ, Permanne R, Koistinen J, Vartiainen T. Polychlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls, naphthalenes, and polybrominated diphenyl ethers in the edible fish caught from the Baltic Sea and lakes in Finland. Environmental Pollution 2006, 141:213– 225. Järemo M, Björklund E, Nilsson N, Karlsson L, Mathiasson L. Utilization of fat retainers in supercritical fluid extraction for the selective extraction of polychlorinated biphenyls from a model fat sample. Journal of Chromatography A 2000, 877:167–180. Jiang Q, Hanari N, Miyake Y, Okazawa T, Lau RKF, Chen K, Wyrzykowska B, So MK, Yamashitab N, Lam PKS. Health risk assessment for polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and dibenzofurans, and polychlorinated naphthalenes in seafood from Guangzhou and Zhoushan, China. Environmental Pollution 2007, 148:31–39. Johansen HR, Rossland OJ, Becher G. Congener specific determination of PCBs in crabs from polluted fjord region. Chemosphere 1993, 27:1245–1252. Juan CY, Thomas GO, Semple KT, Jones KC. Methods for the analysis of PCBs in human food, feces and serum. Chemosphere 1999, 39:1467–1476. Kalantzi OI, Alcock RE, Johnston PA, Santillo D, Stringer RL, Thomas GO, Jones KC. The global distribution of PCBs and organochlorine pesticides in butter. Environmental Science Technology 2001, 35:1013–1018. Kirivanta H, Vartiainen T, Parmanne R, Hallikainen A, Koistinen J. PCDD/Fs and PCBs in Baltic herring during the 1990s. Chemosphere 2003, 50:1201– 1216. Kirivanta H, Ovaskainen E, Vartiainen T. Market basket study on dietary intake of PCDD/Fs, PCBs, and PBDES in Finland. Environment International 2004, 30:923–932. Kitamura K, Takazawa Y, Hashimoto S, Choi JW, Ito H, Morita M. Effective extraction method for dioxin analysis from lipid-rich biological matrices using a combination of pressurized liquid extraction and dimethyl sulfoxide/acetonitrile/hexane partitioning. Analytical Chimica Acta 2004, 512:27–37. Koistinen J, Kirivanta H, Ruokojärvi P, Parmanne R, Verta M, Hallikainen A, Vartiainen T. Organohalogen pollutants in herring from the northern Baltic Sea: Concentration, congeners profiles and explanatory factors. Environmental Pollution 2008, 154:172–183. Korytár P, Danielsson C, Leonards PEG, Haglund P, de Boer J, Brinckman UATh. Separation of seventeen 2,3,7,8- substituted polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like PCBs by comprehensive two-dimensional gas chromatography with ECD. Journal of Chromatography A 2004, 1038: 189–199. Korytár P, Haglumd P, de Boer J, Brinkman UATh. GCxGC for the analysis of organohalogenated microcontaminats. Trends in Analytical Chemistry 2006, 25:373–396.

Kristenson EM, Korytár P, Danielsson C, Kallio M, Brandt M, Mäkelä J, Vreuls RJJ, Beens J, Brinkman UATh. Evaluation of modulators and ECD for comprehensive two-dimensional GC of halogenated organic compounds. Journal of Chromatography A 2003, 1019:65–77. Kristenson EM, Ramos L, Brinkman UATh. Recent advances in matrix solid-phase dispersion. Trends in Analytical Chemistry 2006, 25:96–111. Lehotay SJ. Supercritical fluid extraction of pesticides in foods. Journal of Chromatography A 1997, 785: 289–312. Leonards PEG, Brinkman UATh, Cofino WP. The use of GC with ion trap MS/MS detection for the determination of planar PCBs in biota and sediment. Chemosphere 1996, 32:2381–2387. Letellier M, Budzinski H. Microwave assisted extraction of organic compounds. Analusis 1999, 27:259–271. Liem AKD. Basic aspects of methods for the determination of dioxins and PCBs in foodstuffs and human tissues. Trends in Analytical Chemistry 1999a, 18:429–439. Liem AKD. Important developments in methods and techniques for the determination of dioxins and PCBs in foodstuffs and human tissues. Trends in Analytical Chemistry 1999b, 18:499–507. Llompart M, Pazos M, Landin P, Cela R. Determination of polychlorinated biphenyls in milk samples by saponification-solid-phase microextraction. Analytical Chemistry 2001, 73:5858–5865. Luque de Castro MD, García-Ayuso LE. Soxhlet extraction of solid materials: An outdated technique with a promising innovative future. Analytica Chimica Acta 1998, 369:1–10. Luque de Castro MD, Jimenez-Carmona MM. Where is supercritical fluid extraction going? Trends in Analytical Chemistry 2000, 19:223–228. Malavia J, Santos FJ, Galceran MT. Gas chromatography– ion trap tandem mass spectrometry versus GC-highresolution mass spectrometry for determination of non-ortho-polychlorinated biphenyls in fish. Journal of Chromatography A 2004, 1056:171–178. Manirakiza P, Covaci A, Nizigiymama L, Ntzakimazi G, Schepens P. Persistent chlorinated pesticides and polychlorinated biphenyls in selected fish species from Lake Tanganyika, Burundi, Africa. Environmental Pollution 2002, 117:447–455. Marlowe JL, Knudsen ES, Schwemberger S, Puga A. The AhR displaces p300 from E2F-dependent promoters and repress S-phase specific gene expression. Journal of Biological Chemistry 2004, 279:29013– 29022. Marriott PJ, Haglund P, Ong RCY. A review of environmental toxicant analysis by using multidimensional gas chromatography and comprehensive GC. Chimica Analytica Acta 2003, 328:1–19. Marriott PJ, Shellie R. Principles and applications of comprehensive two-dimensional gas chromatography. Trends in Analytical Chemistry 2002, 21:573– 583. Matthews V, Päpke O, Gaus C. PCDD/Fs and PCBs in seafood species from Moreton Bay, Queensland,

Analysis of PCBs in Food

Australia. Marine Pollution Bulletin 2008, 57:392–402. Mendiola JA, Herrero M, Cifuentes A, Ibañez E. Use of compressed fluids for sample preparation: Food applications. Journal of Chromatography A 2007, 1152:234–246. Mondon J, Nowak BF, Sodergren A. Persistent organic pollutants in oysters and sand flathead from Tasmanian estuarine and costal waters. Marine Pollution Bulletin 2001, 42:157–161. Moon HB, Ok G. Dietary intake of PCDD/Fs and DL-PCBS, due the consumption of various marine organisms from Korea. Chemosphere 2006, 62:1142– 1152. Muir D, Sverko E. Analytical methods for PCBs and organochlorine pesticides in environmental monitoring and surveillance: A critical appraisal. Analytical and Bioanalytical Chemistry 2006, 386:769–789. Müller A, Björklund E, von Holst C. On-line clean up of pressurized liquid extracts for the determination of polychlorinated biphenyls in feedingstuffs and food matrices using gas chromatography–mass spectrometry. Journal of Chromatography A 2001, 925:197– 205. Mullins MD, Pochini CM, McCrindle S, Romkes M, Safe SH, Safe LM. High-resolution PCB analysis: Synthesis and chromatographic properties of all 209 PCB congeners. Environmental Science and Technology 1984, 18:468–476. Nebert DW, Roe AL, Dieter MZ, Solis WA, Yang Y, Dalton TP. Role of AhR and Ah gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochemical Pharmacology 2000, 59:65– 85. Pan J, Yang YL, Xu Q, Chen DZ, Xi DL. PCBs, PCNs and PBDEs in sediments and mussels from Qingdao coastal sea in the frame of current circulations and influence of sewage sludge. Chemosphere 2007, 66:1971–1982. Phillips JB, Beens J. Comprehensive two dimensional gas chromatography: A hyphenated method with strong coupling between the two dimension. Journal of Chromatography A 1999, 856:331–347. Picó Y, Redondo MJ, Font G, Mañes J. SPE on C18 in trace determination of selected PCBs in milk. Journal of Chromatography A 1995, 693:339–346. Priego-Capote F, Luque de Castro MD. Analytical uses of ultrasound-sample preparation. Trends in Analytical Chemistry 2004, 23:644–653. Punín Crespo MO, Lage Yusty MA. Comparison of supercritical fluid extraction and Soxhlet extraction for the determination of PCBs in seaweed samples. Chemosphere 2005, 59:1407–1413. Ramil Criado M, Hernanz Fernández D, Rodríguez Pereiro I, Cela Torrijos R. Application of the matrix solid-phase dispersion to the determination of PCBs in fat by GC-ECD and MS. Journal of Chromatography A 2004, 1056:187–194. Ramos JJ, González MJ, Ramos L. Miniaturized sample preparation of fatty foodstuffs for the determination of PCBs. Journal of Separation Science 2004, 27: 595–601.

47

Ramos L, Eljarrat E, Hernández LM, Rivera J, González MJ. Comparative study of methodologies for the analysis of PCDDs and PCDFs in powdered full-fat milk. PCBs, PCDD, and PCDF levels in commercial samples from Spain. Chemosphere 1999, 38:2577– 2589. Ramos L, Kristenson EM, Brinkman UATh. Current use of pressurized liquid extraction and subcritical water extraction in environmental analysis. Journal of Chromatography A 2002, 975:3–29. Raynie DE. Modern extraction techniques. Analytical Chemistry 2006, 78:3997–4003. Raynie DE. Warning concerning the use of nitrous oxide in supercritical fluid extraction. Analytical Chemistry 1993, 65:3127–3138. Reichard JF, Dalton TP, Shertzer HG, Puga A. Induction of oxidative stress responses by dioxin and other ligands of the AhR. Dose-Response 2005, 3:306–311. Richter BE, Jones BA, Ezzel JL, Porter NL, Avdalovic N, Pohl C. Accelerated solvent extraction: A technique for sample preparation. Analytical Chemistry 1996, 68:1033–1039. Ridgway K, Lalljie SPD, Smith RM. Sample preparation techniques for the determination of trace residues and contaminants in foods. Journal of Chromatography A 2007, 1153:36–53. Rimkus GG, Rummler M, Nausch I. Gel permeation chromatography-high performance liquid chromatography combination as an automated clean up technique for multiresidue analysis of fats. Journal of Chromatography A 1996, 737:9–14. Safe S, Wang F, Porter W, Duan R, McDougal A. AhR agonist as endocrine disruptors: Antiestrogenic activity and mechanisms. Toxicological Letters 1998, 102–103:343–347. Saito K, Sjödin A, Sandau CD, Davies MD, Nakazawa H, Matsuki Y, Patterson Jr DG. Development of an accelerated solvent extraction and gel permeation chromatography analytical method for measuring persistent organohalogen compounds in adipose and organ tissue analysis. Chemosphere 2004, 57:373– 381. Santos FJ, Galceran MT. The application of gas chromatography to environmental analysis. Trends in Analytical Chemistry 2002, 21:672–685. Schantz MM, Bøwadt S, Benner Jr BA, Wise SA, Hawthorne SB. Comparison of supercritical fluid extraction and Soxhlet extraction for the determination of polychlorinated biphenyls in environmental matrix standard reference materials. Journal of Chromatography A 1998, 816:213–220. Schantz MM, Nichols JJ, Wise SA. Evaluation of pressurized fluid extraction for the extraction of environmental matrix reference materials. Analytical Chemistry 1997, 69:4210–4219. Scippo ML, Eppe G, De Pauw E, Maghuin-Rogister G. DR-CALUX screening of food samples: Evaluation of quantitative approach to measure dioxin, furans, and DL-PCBs. Talanta 2004, 63:1193–1202. Seegal RF. Neurochemical effects of co-planar and noncoplanar polychlorinated biphenyls. Neurotoxicology Teratology 1998, 20:349–350.

48

Analysis of Endocrine Disrupting Compounds in Food

Shain W, Bush B, Seegal R. Neurotoxicity of PCBs: Structure-activity relationship of individual congeners. Toxicology and Applied Pharmacology 1991, 111:33–42. Sjödin A, McGahee III E, Focant JF, Jones RS, Lapeza CR, Zhang Y, Wang RY, Patterson Jr DG. Semiautomated high-throughput extraction and cleanup method for the measurement of polybrominated diphenyl ethers and polybrominated and polychlorinated biphenyls in breast milk. Analytical Chemistry 2004, 76:4508–4514. Smedes F, de Boer J. Determination of chlorobiphenyls in sediments-analytical methods. Trends in Analytical Chemistry 1997, 16:503–517. Smith RM. Before the injection: Modern methods of sample preparation for separation techniques. Journal of Chromatography A 2003, 1000: 3–27. Smith RM. Supercritical fluids in separation science: The dream, the reality, and the future. Journal of Chromatography A 1999, 856:83–115. Sparr Eskilsson C, Björklund E. Analyticalscale microwave-assisted extraction. Journal of Chromatography A 2000, 902:227–250. Suchan P, Pulkrabová J, Hajšlová J, Kocourek V. Pressurized liquid extraction in determination of polychlorinated biphenyls and organochlorine pesticides in fish samples. Analytica Chimica Acta 2004, 520: 193–200. Sun C, Dong Y, Xu S, Yao S, Dai J, Han S, Wang L. Trace analysis of dissolved polychlorinated organic compounds in the water of Yangtze River (Nanjing, China). Environmental Pollution 2002, 117:9–14. Tekel J, Hatrìk S. Pesticides residue analysis in plant material by chromatographic methods: Clean up procedure and selective detectors. Journal of Chromatography A 1996, 754:397–410. Tilson HA, Kodavanti PR, Mundy WR, Bushnell PJ. Neurotoxicity of environmental chemicals and their mechanisms of action. Toxicology Letters 1998, 102– 103, 631–635. Thomsen C, Leknes H, Lundanes E, Becher G. A new method for determination of halogenated flame retardants in human milk using solid-phase extraction. Journal of Analytical Toxicology 2002, 26:129–137. Tsang HL, Wu SC, Wong CKC, Leung CKM Tao S, Wong MH. Risk assessment of PCDD/Fs levels in human tissues related to major food items based on chemical analysis and micro-EROD assay. Environmental International 2009, 35:1040–1047. Usydus Z, Szlinder-Richert J, Polak-Juszczak L, Komar K, Adamczyk M, Malesa-Ciecwierz M, Ruczynska W. Fish products available in Polish market: Assessment of the nutritive value and human exposure to dioxins and other contaminants. Chemosphere 2009, 74: 1420–1428. Valsamaki VI, Boti VI, Sakkas VA, Albanis TA. Determination of organochlorine pesticides and PCBs in chicken eggs by matrix solid-phase dispersion. Analytica Chimica Acta 2006, 573–574:195–201. Van Leeuwen SPJ, de Boer J. Advances in the gas chromatographic determination of persistent organic pollutants in the aquatic environment. Journal of Chromatography A 2008, 1186:161–182.

Van Pul WAJ, de Leeuw FAAM, van Jaarsveld JA, van der Gaag MA, Sliggers CJ. The potential for long range transboundary atmospheric transport. Chemosphere 1998, 37:113–141. Verenitch SS, deBruyn MH, Ikonomou MG, Mazumder A. Ion-trap tandem mass spectrometry-based analytical methodology for the determination of polychlorinated biphenyls in fish and shellfish performance comparison against electron-capture detection and high-resolution mass spectrometry detection. Journal of Chromatography A 2007, 1142:199–208. Vives I, Grimalt JO. Method for integrated analysis of polycyclic aromatic hydrocarbons and organochlorine compounds in fish liver. Journal of Chromatography B 2002, 768:247–254. Xiong G, He X, Zhang Z. MAE or saponification combined with microwave-assisted decomposition applied in pretreatment of soil or mussel samples for determination of PCBs. Analytica Chimica Acta 2000, 413:49–56. Weistrand C, Lundèn A, Norèn K. Leakage of polychlorinated biphenyls and naphthalenes from electronic equipment in a laboratory. Chemosphere 1992, 24:1197–1206. Wiberg K, Sporring S, Haglund P, Björklund E. Selective pressurized liquid extraction of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like PCBs from food and feed samples. Journal of Chromatography A 2007, 1138:55–64. Witczak A, Chlewinska M. Two methods of sample preparation for analysis of non-ortho and mono-ortho PCBs congeners in the muscles of selected fish species. Acta Ichthyologica et Piscatoria 2008, 38:63–71. Zajicek JL, Tillitt DE, Schwartz TR, Schmitt C, Harrison RO. Comparison of an enzyme-linked immunosorbent assay (ELISA) to GC: Measurement of PCBs in selected US fish extracts. Chemosphere 2000, 40:539–548. Zhao G, Zhou H, Wang D, Zha J, Xu Y, Rao K, Ma M, Huang S, Wang Z. PBBs, PBDEs and PCBs in foods collected from e-waste disassembly sites and daily intake by local residents. Science of the Total Environment 2009,407:2565–2575. Zohair A, Salim AB, Soyibo AA, Beck AJ. Residues of PAH and PCB and organic pesticides in organicallyfarmed vegetables. Chemosphere 2006, 63:541–553. Zougagh M, Valcárcel M, Rìos A. Supercritical fluid extraction: A critical review of its analytical usefulness. Trends in Analytical Chemistry 2004, 23:399–405. Zuccato E, Fanelli R, Fattore E, Valdicelli L. Levels of PCBs in salmon samples from Europe. Organohalogen Compounds 2004, 66:1998–2001. Zuccato E, Grassi P, Davoli E, Valdicelli L, Wood D, Reitano G, Fanelli R. PCB concentrations in some foods from four European countries. Food and Chemical Toxicology 2008, 46:1062–1067. Zuccato E, Calvarese S, Mariani G, Mangiapan S, Grasso P, Guzzi A, Fanelli R. Level, source, and toxicity of PCBs in the Italian diet. Chemosphere 1999, 38:2753–2765.

Chapter 3 Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food Luisa R. Bordajandi, Belén Gómara, and María José González

Introduction Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are a group of toxic and highly persistent organic compounds that consist of 210 congeners. They have never been deliberately produced, but they have been accidentally formed as byproducts of a wide variety of chemical industries and combustion processes that contain chlorine and chlorinated aromatic hydrocarbon sources (Fiedler 2007). They are of great concern due to the extreme toxicity of the 2,3,7,8 chlorine-substituted congeners and their presence in all compartments of the environment. Due to their low water solubility, hydrophobicity, and resistance to degradation, these substances are found in a wide range of biological samples and tend to accumulate in animal and human adipose tissues through the food web (Ormerod et al. 2000). PCDDs and PCDFs with the 2,3,7,8 configuration (17 congeners) are the most toxic among those studied in experimental animals. They are a potential human carcinogen and disrupt the immune and endocrine systems, producing many adverse effects in humans and wildlife (van Leeuwen et al. 2000; Lundgren et al. 2002; van den Berg et al. 2006; Lin et al. 2007). Recently, there has been heightened concern regarding the role of PCDD/PCDFs Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

as estrogenic substances, which contribute to the development of many adverse effects in humans and wildlife. The growing number of reports showing that environmental contaminants, including PCDD/PCDFs, possess estrogenic activity has led to a working hypothesis that adverse trends in male reproductive health may be, at least in part, associated with exposure to estrogenic and other hormonally active environmental chemicals during fetal development and childhood. The presence in the environment of contaminants that act by disrupting homeostasis of sex hormones is of growing concern. Most attention so far has been given to chemicals such as PCDD/PCDFs, which have endocrine disruptor properties through aryl hydrocarbon receptor (AhR) mediation, are structurally related to natural estrogens, and have affinity to the estrogen receptor. Ingestion of contaminated food is the major route of human exposure to these compounds, accounting for more than 95% of the exposure, with inhalation and dermal contact accounting for the rest (Sweetman et al. 2000; Hays and Aylward 2003). Public concern over the adverse health effects of these toxicants has been intensified in the last few years by a number of dioxin contamination incidents involving food and feedstuffs (Malisch 2000; Bernard et al. 2002; Hoogenboom et al. 2007). Reports concerning toxicological aspects have led to a reevaluation of the tolerable daily intake (TDI) of dioxins (van Leeuwen et al. 2000) and have prompted wide-ranging efforts and 49

50

Analysis of Endocrine Disrupting Compounds in Food

Table 3.1. Maximum levels and action levels of the sum of PCDDs and PCDFs in foodstuffs. Foodstuffs Meat and meat products (excluding edible offal) of the following animals: Bovine animals and sheep Poultry Pigs Liver of terrestrial animals and derived products Muscle meat fish, fishery products, and products thereof Raw milk and dairy products, including butterfat Hen eggs and egg products Fats and oils Fat of the following animals: Bovine animals and sheep Poultry Pigs Mixed animal fats Vegetable oils and fats Marine oil for human consumption

Maximum Levels (pg/g fat)

Action Levels (pg/g fat)

3 2 1 6 4a 3 3

2 1.5 0.6 4 3a 2 2

3 2 1 2 0.75 2

2 1.5 0.6 1.5 0.5 1.5

a

pg/g fresh weight Levels expressed as World Health Organization (WHO) toxic equivalents (WHO-TEQ) of PCDD/PCDFs per gram of lipid weight using the WHO-toxic equivalency factors (WHO-TEFs) for human risk assessment based on the conclusions of the WHO meeting in Stockholm, Sweden, 15 to 18 June 1997 (van den Berg 1998, Commission Regulation 1881/2006/EC, Commission recommendation 201/2002/EC).

the tightening of regulations to reduce dioxin release into the environment (Commission Regulation 76/2000/EC). To prevent the health risk from dioxin exposure, the European Commission has recently established maximum permissible levels of dioxins and dioxin-like polychlorinated biphenyls (PCBs) in foods (Council Regulation 1881/2006/EC). Table 3.1 shows the maximum levels allowed for various foodstuffs, such as meat, fish, dairy products, and vegetable and fish oils, that are applicable for foods containing more than 1% fat. The lower value, 0.75 pg of toxic equivalents (World Health Organization PCDD/PCDF toxic equivalents, or WHOTEQs) per gram of fat weight, is assigned to vegetable fats and oils, whereas the maximum level is for fish and seafood (4 pg of WHOTEQs) per gram of fresh weight). There are large amounts of data in the literature concerning PCDD/PCDF levels in foodstuffs from different countries (Hayward et al. 2001, Focant et al. 2002a; Knutzen et al. 2003; Kiviranta et al. 2004; Papadopoulus et al. 2004; Fernández et al. 2004; Baars et al. 2004; Sasamoto et al. 2006; El-Kady et al.

2007; Llobet et al. 2008) including, in most of them, all food categories of animal origin. Some of these studies showed a temporal decreasing trend of the PCDD/PCDF content over the years (Knutzen et al. 2003; Sasamoto et al. 2006; Llobet et al. 2008), making the current dioxin levels at sub-parts per billion levels and rendering their analysis complex and challenging. Usually, the analysis of these compounds in foodstuffs requires complicated and very time-consuming sample extraction and cleanup procedures, followed by gas chromatography (GC) separation and identification and confirmation by HRMS (high-resolution mass spectrometry) (Liem 1999a, b). Several extraction and cleanup procedures are described in the literature, and which is chosen depends on individual analytical laboratories. The most used techniques are Soxhlet (Abad et al. 2002; Papadopoulus et al. 2004; Llobet et al. 2008); solid-phase extraction (SPE) (Focant et al. 2003); matrix solid-phase dispersion (MSPD) (Santillo et al. 2003; Fernández et al. 2004; Bordajandi et al. 2004; Gómara et al. 2005); liquid–solid extraction

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

(LSE) (Mayer 2001; Jacobs et al. 2002; Knutzen et al. 2003), including acid and basic digestion (Tsutsumi et al. 2001; Fernandes et al. 2004; Sasamoto et al. 2006); and pressurized liquid extraction (PLE) (also called accelerated solvent extraction, or ASE) (Focant et al. 2001; Bernsmann and Fürst 2004; Grümping et al. 2008; Lund et al. 2008; Traag et al. 2008). Cleanup procedures, including open column chromatography on activated Florisil, alumina, silica, and carbon, and size-exclusion chromatography are also currently used (Tsutsumi et al. 2001; Mayer 2001; Jacobs et al. 2002; Knutzen et al. 2003; Otaka and Hashimoto 2004; Papadopoulos et al. 2004; Bordajandi et al. 2004; Baars et al. 2004; Sasamoto et al. 2006; Llobet et al. 2008). Automatic online cleanup procedures, such as the Power-Prep system, which combines the extraction and cleanup step to obtain extracts ready for GC analysis with the maximum extraction efficiency and to overcome matrix-related interferences, are increasingly used as a routine method (Focant et al. 2001; Abad et al. 2002; Grümping et al. 2008; Lund et al. 2008; Traag et al. 2008). Over the last few years, the European Union (EU) has initiated a large-scale research project to develop new analytical methodologies for the determination of dioxins (most of them including dioxin-like PCBs) in food matrices to serve as alternatives to GCHRMS. This last technique is considered the benchmark for accurate and specific determination of these compounds in food samples as described in the Environmental Protection Agency (EPA) and EU official methods (U.S. EPA 1999, 2002; Commission Regulation 1883/2006/EC). GC-HRMS provides enough specificity and selectivity at concentration levels down to fentograms per gram for the analysis of these compounds, but it is a relatively expensive technique and requires qualified personnel to run the tests. As a result, alternative techniques, such as GC coupled to ion trap mass spectrometry (GC-ITMS), working in tandem mode (MS/MS) (Malavia et al. 2007a, b; 2008), as well as comprehen-

51

sive two-dimensional GC (GCxGC) coupled to microelectron-capture detection (μ-ECD) (Danielsson et al. 2005) and time of flight (TOF) MS (Focant et al. 2005b; Hoh et al. 2008), have recently been validated for acceptance as an alternative to GCHRMS. In addition, bioanalytical methods have improved considerably in sensitivity and selectivity to the extent that they can be used as screening methods to determine the total quantities of dioxin-like compounds (Hoogenboom et al. 2006). Following a review on the analytical methods for the determination of PCDD/ PCDFs in fish and seafood products recently published by Bordajandi et al. (2010), this chapter focuses on the review of the analytical methods for the analysis of dioxins and furans in food and food products. Although almost all studies include dioxin-like PCBs, we specifically focus on those targeting the 17 toxic 2,3,7,8-PCDD/PCDFs in food and food products. Attention has been paid to both methods that are in current use and methods that have recently been developed for each step of the analysis from sample preparation to instrumental determination of these congeners.

Sample pretreatment and recovery studies Sample storage and treatment Food and food products are usually preserved by freezing immediately, either in the field or at the laboratory. Whenever possible, the solid food samples (meat, eggs, fish) should be dissected immediately and the tissues stored in individual packs of the size required for analysis to minimize thawing of subsampling material. Meat, eggs, and fish tissues are first macerated and then freeze-dried, or alternatively ground with sodium sulphate and silica to reduce the water content and rupture cell walls. These are the two most commonly used pretreatment procedures for these types of matrices (Table 3.2). Liquid samples such as milk and yogurt can be frozen by layers

52 Homogenized, frozen before freeze-drying, and grinding Homogenized, frozen before freeze-drying (a, b) Dissolved in hexane (c)

a

a, b, c

Skinned (a), homogenized and freeze-dried (a, b) Centrifugation and freeze-dried (c)

Skinned (a), homogenized and freeze-drying (a–c) Dissolved in n-hexane (d, e) Skinned, homogenized, and frozen before freeze-drying Skinned (a), homogenized (a–f), addition of solvents (a–c, f), no treatment (d–e) Skinned (a), blended and freeze-dried Skinned (a), homogenized and frozen before freeze-drying (a–c) No treatment (d, e) Skinned, homogenized and ground with sodium sulfate Skinned, homogenized and ground with sodium sulfate Skinned (a), homogenized and blended (a–c) No treatment (d, e)

Sample Preparation

a–c

a–c, f

a

a

a–f, h

a–c, f

a–f (cooked and fresh)

a

a–h

Type of Fooda

ASE

ASE

LSE

LLE (pretreatment with alkali hydrolysis)

LSE

LSE

LSE (hydrolysis as previous step) MSPD

LLE

MSPD

Soxhlet (a–c) No extraction (d, e)

Extraction

Multilayer silica gel (neutral, acid, basic) Silica gel and alumina

Multilayer silica gel (neutral, acid, basic)

Multilayer silica gel (neutral, acid, basic and AgNO3) and alumina Charcoal and Florisil

Active carbon

Graphitic carbon

Active carbon

GPC (up to 4 g of Automated online Power-Prep system lipids) using LCDS, LCDA, and LCDC Automated online Power-Prep system using HCDS,c LCDS, LCDA, and LCDC

Focant et al. 2001

Grümping et al. 2008

After extraction After extraction

Mayer 2001

Tsutsumi et al. 2001

Jacobs et al. 2002

Knutzen et al. 2003

Fernandes et al. 2004 Bordajandi et al. 2004

Chen et al. 2008

Gómara et al. 2005

Llobet et al. 2008

Reference

After extraction

Prior extraction

After extraction

Prior extraction

Prior extraction Prior extraction

Prior extraction

Florisil column Active carbon Graphitic carbon

Prior extraction

Prior extraction

Addition of ISSb

Graphitic carbon

Alumina

Fractionation

GPC chromatography or multilayer silica gel (neutral, acid, basic) Automated online Power-Prep system using HCDS,c LCDS, LCDA, and LCDC

H2SO4 concentrated

Multilayer silica gel (neutral, acid, basic) and alumina

Multilayer silica gel (neutral, acid, basic)

Silica gel modified with H2SO4 (22% and 44%)

Multilayer silica gel (neutral, acid, basic)

Silica gel modified with H2SO4 (22% and 44%) H2SO4 concentrated

Chemical Interferences Elimination Multilayer silica gel (neutral, acid, basic)

Lipid Elimination

Cleanup

Table 3.2. Sample preparation (extraction and cleanup steps) for dioxin analysis in food samples (2001–2008).

53

Blended, homogenized and ground with sodium sulphate (a–c) No preparation (d, e) Homogenized and freeze-dried

a–e

Homogenized, addition of sodium oxalate and methanol Homogenized and addition of potassium oxalate and acetonitrile Freeze-dried No preparation No preparation

c

d

e

e

a–d e

c

b

a–e

a

a–c

Homogenized, frozen before freeze-drying (a–c, f, h) No treatment (d) Homogenized and freeze-dried Skinned, homogenized and freeze-dried Homogenized and freeze-dried Homogenized

a–d, f, h

Static and dynamic dialysis ASE No extraction

No extraction

MSPD

Active carbon

Active carbon

Multilayer silica gel (neutral, acid, basic) Active and alumina carbon Multilayer silica gel (neutral, acid, basic). Active Basic alumina and C18 carbon Automated online Power-PrepT system H2SO4 concentrated using LCDS, LCDA, and LCDC Automated online Power-Prep system using LCDS, LCDA, and LCDC Active carbon (reflux unit)

Multilayer silica Alumina gel (neutral, acid, basic) Multilayer silica gel (neutral, acid, basic)

Alumina

Active carbon

Alumina Multilayer silica gel (neutral, acid, basic and modified with AgNO3) Active carbon (reflux unit)

Fractionation

Alumina

Chemical Interferences Elimination

Multilayer silica gel (neutral, acid, basic and modified with AgNO3)

Lipid Elimination

Cleanup

Automated online Power-Prep system using HCDS,c LCDS, LCDA, and LCDC

Semiautomated online system using multilayer silica (neutral, acid, basic), Florisil and active carbon GPC (SX3) for Graphitic carbon lipid removal Alumina and silica gel HPLC (PYE)

Automated online Power-PrepT system using C18, LCDS, and LCDC

LLE

ASE

Soxhlet

ASE

Soxhlet

MSPD

No extraction (d, e) Soxhlet

Soxhlet

LLE (pretreatment with alkali hydrolysis) LLE (pretreatment with alkali hydrolysis) LLE

Extraction

Prior extraction Prior extraction

Prior extraction Prior GPC

Prior extraction

Prior extraction Prior extraction Prior extraction Prior extraction Prior extraction

Prior extraction

Prior extraction

Prior extraction

Prior extraction

Prior extraction

Addition of ISSb

b

(a) fish and sea fish, (b) meat and meat products, (c) dairy products, (d) vegetable oil and butter, (e) fish oil, (f) eggs, (g) vegetables and fruits, (h) cereals and pulses. Addition of 13C12-labeled internal standards (known amount). c HCDS, high-capacity disposable silica: multilayer silica (28 g of acid, 16 g of basic, and 6 g of neutral) column, to remove large amounts of fat. LCDS, low-capacity disposable silica; LCDA, low-capacity disposable alumina; LCDC, low-capacity disposable active carbon.

a

Homogenized, frozen before freeze-drying

a–f g, h

a–d, f, h

Homogenized and frozen at −20°C

Sample Preparation

a, b

Type of Fooda

Traag et al. 2008

Hess et al. 2001

Santillo et al. 2003 Hoh et al. 2008

Focant et al. 2003

Wittsiepe et al. 2001 El-Kady et al. 2007 Abad et al. 2002 Lund et al. 2008 Baars et al. 2004

Fernández et al. 2004

Kiviranta et al. 2004

Papadopoulos et al. 2004

Otaka and Hashimoto 2004 Sasamoto et al. 2006

Reference

54

Analysis of Endocrine Disrupting Compounds in Food

and freeze-dried, or kept cold at −20°C until the extraction procedure, or pretreated with acetonitrile and potassium oxalate or with the mixture ethanol-hexane-acetone (Table 3.2). Other liquid samples, such as oils and butter samples, are usually dissolved in different kinds of solvents and directly submitted to the cleanup procedure. Alternatively, they are kept cold until the extraction procedure by dialysis (Table 3.2). It should be noted that the concentration of the 2,3,7,8 PCDD/PCDF congeners is generally at the amount of fentogram per gram. It is therefore necessary to analyze samples containing around 6 g of fat, which requires large amounts of fresh sample: around 600 g for bivalves (1% fat), 200 g for cow’s milk and dairy products (3% fat), 30– 40 g for salmon (20% fat), and 6 g of butter and oil (100% of fat). In any case, this amount is much higher than that usually required for the analysis of other persistent organic pollutants (POPs). For this reason, almost all sample pretreatment methods involve freezedrying, which eliminates the water content and drastically reduces the sample size. On the other hand, the freeze-drying step takes 48 hours, which considerably increases the total analysis time.

Spiking and recovery studies The analysis of the 17 target PCDD/PCDFs in food samples requires the use of internal standards for accurate quantification and for method performance check. Isotope dilution mass spectrometry (IDMS), that is, the addition of labeled 2,3,7,8-PCDD/PCDF standards, is the most elegant way to overcome the whole problem of quantification and allows the recovery calculation of the whole procedure (extraction + cleanup + analytical determination) (U.S. EPA 2002; Commission Regulation 1883/2006/EC). Prior to extraction, the seventeen 13C12 2,3,7,8-PCDD/ PCDF–labeled isotopes are added to the sample at a known concentration as extraction standards for quantification (Table 3.2). Two more 13C12-PCDD congeners (1,2,3,4-

TCDD and 1,2,3,7,8,9-HxCDD) are added as instrumental standards to the extract at a known concentration prior to the instrumental determination. The ratio of the labeled and native compounds is measured by MS and automatically accounts for any losses in the procedure. Although it is not necessary to calculate the recoveries for quantification purposes, they are calculated as a quality parameter to check the performance of the method from the ratio of the labeled congeners in the extraction and recovery standards. Although most of the methodologies in PCDD/PCDF food analysis are usually used to spike the samples with the labeled standards before the extraction step, other methodologies spike the fatty extract obtained after the extraction step (Table 3.2).

Extraction techniques The purpose of the extraction step is to remove the bulk of the sample matrix and to transfer the fraction containing the analytes to a suitable solvent. Extraction techniques for food and food products are generally based on the assumption that lipophilic compounds such as PCDD/PCDFs predominantly occur in the fat fraction of the food matrix. Therefore, they are based on general methods for isolation of the lipid fraction from the sample matrix (Tables 3.2 and 3.3). Conventional extraction methods are Soxhlet (SOX), solid-phase extraction (SPE), matrix solid phase dispersion (MSPD), and supercritical fluid extraction (SFE) (van Babel et al. 1996; van der Velde et al. 1996). For liquid samples, some authors are still using liquid– liquid extraction (LLE) for milk samples and dialysis for oils (vegetable and fish). Although Soxhlet extraction is a longstanding and proven technique used frequently for many years, the need to change the nature of the solvent (e.g., to avoid the use of chlorinated solvents), the large amounts of solvents required, and the long extraction times have driven the development of alternative extraction techniques in recent years.

55

Homogenised with silica gel:anhydrous sodium sulphate (1:4, w:w) No

MSPD

LLE (liquid samples)

LLE (including acid/basic hydrolysis)

2 M KOH 2 M KOH:ethanol 1 M KOH Acid hydrolysis Ethanol:acetone: n-hexane (1:1:1) Sodium oxalate and methanol Sodium oxalate and methanol Ethanol

No

ASE

LSE (solid samples)

No

Pretreatment

Soxhlet

Technique

Diethyl ether and petroleum ether AOAC International method n-hexane

Acetone:pentane (12:88) Cyclohexane:DCM (80:20) Acetone:n-hexane (1:1) Acetone:n-hexane (1:1) Acetone:n-hexane (1:1) n-hexane:acetone (2:1) n-hexane:DCM:2isopropanol Cyclohexane:DCM (1:1) n-hexane n-hexane n-hexane n-hexane n-hexane

Toluene/2 methoxy ethanol (9:1) Toluene Toluene Toluene:acetone Toluene n-hexane Cyclohexane n-hexane/acetone (75:25) n-heptane n-hexane:acetone (7:3) n-hexane:DCM:methanol

Solvent Used

≅200

No No

Not indicated >12 h

Room temperature Room temperature

No

>14 h Room temperature

≅200

No No No No No No

>3 h 12 h 1h 2h Not indicated 2h Room temperature Room temperature 70°C (hydrolysis) Room temperature Room temperature Room temperature

650 ≅180 ≅70 ≅180 Not included 400

Yes Yes Yes No No

No Yes

No No No No No Yes No Yes No No

No

Including Fat Retainer

6h 6h 6h Not indicated Not indicated

Room temperature Room temperature Room temperature Room temperature Room temperature

18 h 24 h 18 h 24 h 15 min 30 min 30 min 20 min 30 min 20 min

Room temperature Room temperature Room temperature Room temperature 1500 psi 120°C; 1500 psi 120°C; (12 MPa) 100°C; FV 60% 100°C; 1500 psi 100–185°C; 1500 psi 100°C Room temperature 20 min 6h

24 h

Extraction Time

Room temperature

Extraction Conditions

≅400 ≅400 ≅400 Not included Not included

≅200 330

≅40 ≅150 ≅100 ≅100 ≅150 ≅200

≅300

Amount of Solvent (mL)

Chen et al. 2008

Papadopoulus et al. 2004

Baars et al. 2004

Knutzen et al. 2003 Tsutsumi et al. 2001 Otaka et al. 2004 Sasamoto et al. 2006 Fernandes et al. 2004 Chen et al. 2008

Bordajandi et al. 2004 Fernández et al. 2004 Gómara et al. 2005 Mayer 2001 Jacobs et al. 2002

Lund et al. 2008 Santillo et al. 2003

Abad et al. 2002 Kivirinanta et al. 2004 Papadoupoulus et al. 2004 Bocio and Domingo 2005 Focant et al. 2001 Bernsmann and Fürst 2004 El-Kady et al. 2007 Wiberg et al. 2007 Traag et al. 2008 Grümping et al. 2008

Wittsiepe et al. 2001

References

Table 3.3. Comparison of the experimental conditions of the most used extraction techniques for dioxin analysis in foodstuffs of animal origin.

56

Analysis of Endocrine Disrupting Compounds in Food

Substantial progress has been made toward developing enhanced extraction techniques such as microwave-assisted solvent extraction (MASE) (Camel 2000) and, especially, the widely used pressurized liquid extraction (PLE or ASE, accelerated solvent extraction, named after the PLE system marketed by Dionex). Saponification under alkaline and acidic conditions followed by extraction with organic solvents is often employed for the analysis of large amounts (up to 100 g) of fat. However, that method is known to lead to degradation of dioxins in proportion to their chlorine content, and in the case of PCDFs, to the production of artifacts such as lower chlorinated PCDFs and ethoxy-PCDFs (Ryan et al. 1989). Finally, it is worth noting that although the approach has not yet been studied extensively, some applications have already demonstrated the potential of sonication (ultrasound-assisted extraction, or USE) for food dioxin analysis (Lanbropoulou et al. 2006; Ahn et al. 2006).

Soxhlet extraction SOX is one of the most frequently used liquid– solid extraction methods, developed in the late nineteenth century and still routinely used for extraction of dioxins from food samples (Tables 3.2 and 3.3). However, the technique has a number of drawbacks, among them the large volume of solvent required (200 mL for 100 g of tissue), the long extraction time (more than 18 hours), the generation of dirty extracts that require extensive cleanup, and the impossibility of automation. In order to overcome these drawbacks, alternative extraction strategies have been developed, offering analysts a choice of newer techniques such as SPE, MSPD, and more recently, PLE (or ASE).

Solid-phase extraction and matrix solid-phase dispersion SPE is today a classic extraction system, thanks mainly to the popularization of SPE

cartridges, which have been successfully applied to biological human fluids (Chang et al. 1993) and liquid food samples (cow’s and breast milk samples) using C18-bound silica phase (Focant et al. 2003). However, in the case of solid samples, SPE is less popular and has almost never been used to extract dioxins from food and food products because of the large amount of sample needed. To the contrary, MSPD, using open conventional glass chromatography columns, is very often used in routine analysis of food samples (Tables 3.2 and 3.3). In MSPD, the sample is mixed or blended with an appropriate sorbent (e.g., C18, silica) until a homogeneous mixture is obtained. This mixture is then packed into a glass column from which the analytes of interest are eluted with a suitable organic solvent. The extraction and first cleanup steps are performed at the same time, and most of the artifacts are eliminated. Because a large amount of sample is needed, the method compares unfavorably with SOX in terms of the amount of solvent required (around 400 mL).

Supercritical fluid extraction SFE is another classic method for food dioxin analysis, but it is not as popular as SOX and MSPD. SFE has attracted intense interest during the past 20 years, mainly for extraction of solid samples, because it offers short extraction times and minimal use of organic solvents (Smith 1999; Ahmed 2003). Carbon dioxide (CO2) is mostly used as the extraction solvent because of its moderate critical temperature (31°C) and pressure (73 atm). In the 1990s, SFE instruments became commercially available, enabling larger sample sizes and rendering it more suitable for wider applications. For food matrices, fat retainers such as Florisil and silica are usually introduced in the extraction thimble to achieve a fat-free extract. Some applications for fatty food dioxin determinations were published several years ago (van Babel et al. 1996; van der

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

Velde et al. 1996), but currently the focus has been on environmental samples such as soils and sediments (Miyawaki et al. 2008). Although SFE extraction is automated and offers a short extraction time and minimal use of organic solvents with no additional cleanup step before GC-MS, it is not widely used because of the large number of parameters that have to be optimized, especially in the analyte collection chamber, and the high cost of the equipment. Like SPE, the use of SFE for dioxin analysis is not common due to the large amounts of sample required (5–10 g of lipid equivalents) to be able to reach the low levels at which dioxins are present in food samples.

Pressurized liquid extraction One of the most recent solid-sample extraction methods used as an alternative to classic extraction techniques such as SOX and MSPD is PLE or accelerated solvent extraction (ASE). Unlike SFE, an organic solvent replaces CO2, and increased pressures and temperatures are used to speed up the extraction procedure. PLE uses conventional liquid solvents at high pressures (1500 psi) and temperatures (100°–120°C) to extract solid samples quickly and with much less solvent than conventional techniques. Food samples are placed in extraction cells that are filled with an extraction solvent and then heated. The sample is statically extracted for 5–10 min, with the expanding solvent vented to a collection vial. Following this period, compressed nitrogen is used to purge the remaining solvent into the same vial. The entire procedure is completed in 15–30 min per sample and uses only 40–200 mL of solvent. The main disadvantage of PLE (or ASE) is that sample cleanup is still necessary. For this reason, applications dealing with selective extraction procedures, where integrated cleanup strategies are used to combine extraction and cleanup or fractionation to further simplify all the sample-preparation

57

steps, are of special interest. The first online cleanup attempt was performed for the extraction of PCBs from foodstuffs by using acidic alumina in the extraction cell (Dionex Application Note 322 1996). Other fat retainers, for example, basic alumina, neutral alumina, acidic alumina, Florisil, and sulfuric acid–impregnated silica, also were tested for PCB extraction (Björklund et al. 2001). Given the good results obtained, this approach was also applied to the extraction of dioxins. Bernsmann and Fürst (2004) first described the extraction of PCDD/PCDFs and PCBs from feeding stuffs using sulfuric acid– impregnated silica as a fat retainer with very good results. In this study, the integrated cleanup approach was found to be equivalent to SOX extraction. As part of the DIFFERENCE project, Wiberg and coworkers (2007) evaluated traditional extraction techniques versus alternative techniques such as PLE (or ASE) for PCDD/PCDF and dioxin-like PCB determinations in food and feed, including certified reference materials. They demonstrated that PLE is more of a quantitative extraction process than other conventional techniques. The PLE method, in combination with HRMS detection, meets the quality criteria for official control of dioxins in foodstuffs (Commission Regulation 1883/2006/EC). One of the last developments is the combination of PLE with integrated carbon fractionation (Nording et al. 2005), in which dioxins can be fractionated and obtained in backward elution, and only a small, miniaturized, multilayer silica column cleanup is required after PLE and before instrumental determination. Some attempts have also been made to combine PLE with automated cleanup systems, in particular the Power-Prep system (which is discussed below in the section on cleanup), to construct a fully automated system (extraction plus cleanup). However, the results have not been satisfactory; in this type of combination, PLE is used as a dynamic system and requires the incorporation of a

58

Analysis of Endocrine Disrupting Compounds in Food

concentration phase prior to Power-Prep, rendering automation virtually impossible and considerably increasing the analysis times (Focant et al. 2005a). At present, the PLE system is working in static mode, but the HPLC system (pump and valves) is located in an individual module separated from the low-pressure module and can operate at pressures up to 2500 psi (Focant et al. 2005a). More extensive information about this technique can be found in the literature, where there are some reviews dealing with PLE for POPs in foods (Björklund et al. 2006; Wiberg et al. 2007) and biological matrices (Focant et al. 2004). Tables 3.2 and 3.3 show some examples in which PLE has been used as a routine method in the analysis of dioxins in foods with and without the use of a fat retainer in the cell extraction.

Microwave-assisted solvent extraction MASE has been only recently introduced for the analysis of dioxins. To our knowledge, there are no published studies in which MASE was used for dioxin food extraction. In recent years, this technique has attracted growing interest because it allows rapid extraction of solutes from solid samples by employing microwave energy as a source of heat, with extraction efficiency comparable to that of classic techniques. The partitioning of the analytes from the sample matrix to the later extractant depends on the temperature and the nature of the extractant. Unlike conventional systems, microwaves heat the entire sample simultaneously without heating the vessel; thus, the solution reaches its boiling point very rapidly and the extraction time is very short (Camel 2000). In view of the good results of MASE in the extraction of PCBs and DDTs (Vetter et al. 1998; de Boer 1988) from biological tissues and dioxins from environmental samples (Eljarrat et al. 1998; Miyawaki et al. 2009), this technique is very attractive for dioxin analysis in food samples. Its main drawbacks are the loss of more vola-

tile solutes if the temperature rises rapidly and the need for the solution to be cooled to room temperature after extraction before the vessels can be opened, which increases the overall extraction time. In addition, it is not possible to automate the procedure to incorporate cleanup steps.

Comparison of extraction techniques Table 3.3 shows the experimental conditions and benefits of the extraction methods currently used in most literature. The data presented in Table 3.3 show that PLE (or ASE) with a fat retainer in the extraction chamber compares favorably with conventional classical extraction techniques for dioxin analysis in foodstuffs. The PLE extraction technique drastically reduces the amount of solvent used and the analysis time, mainly because extraction and primary cleanup could be automatically performed in one step within 90 min. Other advantages of this method are the possibility of working with six or more extractions in parallel and, in the future, the online automation including cleanup steps.

Cleanup methods Analytical procedures for the determination of PCDD/PCDFs in food samples involve sophisticated and tedious cleanup methods. Several steps are usually required to remove the bulk of coextractants (including lipids) in order to finish with an extract containing only PCDD/PCDFs in which the analytes can be detected at the ultratrace levels at which they occur in food samples. The choice of a particular sequence of steps will depend very much on the analytical system that is used. Sample extraction, cleanup, and GC method together form a delicately balanced combination, each part contributing to the ultimate specificity and selectivity. For the determination of dioxins, nearly all established schemes involve combinations of cleanup methods originally developed for the analysis of PCBs

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

59

and organochlorinated pesticides (OCPs) (solid–liquid adsorption chromatography using Florisil, silica and alumina, gelpermeation chromatography [GPC], and HPLC) in combination with an active carbon step to isolate the specific fraction containing the dioxins without chemical interference (Table 3.2). In many of the methods used today, the sample extraction and cleanup steps are combined online or at-line, and some are automated.

used for dioxin and other POP analysis in a large variety of fatty food samples such as oils and butters (Strandberg et al. 1998, Hess and Wells 2001; Grochowalski and Wojtalewicz 2005; Gómara et al. 2006). Although it is efficient, simple, versatile, and does not entail excessive solvent use, this procedure is not often used for dioxin analysis because it is time consuming (72 h). In addition, online coupling, either with extraction or with cleanup steps, is not possible.

Lipid removal

Isolation of uncommon chemical interferences

Lipid removal is the first step in the cleanup process, where usually other interference is also eliminated. Several different methods have been used to remove lipids and similar compounds in POP food analysis (Table 3.2), including destructive methods (e.g., sulfuric acid, silica modified with sulfuric acid, Florisil, and alumina) and nondestructive methods (e.g., GPC and dialysis). GPC, which is sometimes referred to as sizeexclusion chromatography (SEC), has been successfully applied to food samples for POP analysis using SX-3 Biobeds (200–400 mesh) in a range of column sizes and solvents. It can be fully automated and, unlike adsorption chromatography, it is also more suitable for the isolation of unknown contaminants on whose polarity or chemical functionality there is little information. The method can also handle a large mass of lipid in each sample (e.g., columns of about 500 × 25 mm ID can handle up to 500 mg of lipids) compared to adsorption columns that are limited to 50 mg of lipids per gram of adsorbent (Ahmed 2001; de Boer and Lau 2003; Hoh et al. 2008). Dialysis with semipermeable membranes (SPMs) in an organic solvent can separate other similar POPs from lipids. The method can eliminate more than 20 g of lipids in a single membrane with acceptable recoveries of internal standards, practically irrespective of the amount and type of lipid dialyzed. The method has been successfully

For dioxin analysis in a food matrix, an additional purification step is necessary to eliminate other interference (including any other lipids). A combination of adsorbents (neutral, basic and acid alumina, silica gel, modified silica gel with acids and basics, and AgNO3, C18, carbon, and Florisil, in multilayer or onelayer columns) and solvents with different polarities and dielectric constants are used to eliminate interference (Table 3.2). It is wellknown to experts that the application of the extract to a strongly basic adsorbent (potassium or cesium hydroxides) silica gel with a low-polarity solvent, for example, hexane, is very effective for removing trace residues of acidic compounds such as phenolic and carboxylic acids and sulfonamide compounds (Smith et al. 1984). On the other hand, sulfuric acid–impregnated silica gel (20–40% w/w) is very effective in removing numerous types of compounds by dehydration, acidcatalyzed condensation, and oxidation reactions (Lamparski et al. 1979). Alumina (basic, acid, and neutral), silica gel modified with AgNO3, and Florisil are used at different activation grades, mainly to eliminate all other lipids and other coextractants (Ramos et al. 1997; Liu et al. 2006). The literature gives no indication of preferences for any specific adsorbent or solvent, the choice of which depends more on the laboratory’s preferences than on performance. Cleaning up food

60

Analysis of Endocrine Disrupting Compounds in Food

samples for dioxin analysis is a laborious and tedious task, which has to be validated. The combination of adsorbents and solvents chosen to obtain a clean extract without any dioxin loss before the GC-MS analysis is up to each laboratory.

Fractionation/group separation Normally, a group separation is necessary before final analysis of dioxins by GC-HRMS. At this stage, the cleaned extract may contain other similar organohalogen compounds such as PCBs. With the exception of non-ortho PCBs, dioxins are present at substantially lower concentrations than the other POPs, and it is therefore necessary to separate dioxins from the bulk of POPs. The methods available for the isolation of POPs into separate fractions prior to GC analysis are based on the spatial planarity of dioxins, which are used to separate them as a distinct fraction. The available methods for fractionation have been extensively reviewed (Hess et al. 1995; Concejero et al. 2001). Open liquid chromatography columns of Florisil, alumina, active carbon, and graphitic carbon are among the most widely used methods (Table 3.2). HPLC with either porous graphitic carbon (PGC) (Creaser and Al-Haddad 1989; de Boer et al. 1993), active carbon (Lundgren et al. 2002), or 2-(1-pyrenyl)ethyldimethylsilylated silica gel (PYE) columns (Ramos et al. 1999; Hess et al. 1995; Díaz-Ferrero et al. 2005) do not have an extended use despite the inherent advantages of HPLC. Concejero and coworkers (2001) studied the feasibility of different adsorbents. Two active carbons (Amoco PX-21 and Carbosphere), two graphitic carbons (Carbopack B and C), and one HPLC stationary phase, PYE (typically used for PCB and PCDD/ PCDF fractionation), were studied for fractionation of PCBs and PCDD/PCDFs. Recoveries for fractionation of the target compounds with all the sorbents studied were generally good, and the reproducibility was

satisfactory. All were able to isolate PCDD/ PCDFs from PCBs, which could interfere in the final determination of the PCDD/PCDFs by GC-HRMS. As a result, Carbopack B (as SPE cartridges) and PYE (as HPLC column) were considered the most valuable alternatives for simultaneous fractionation of PCDD/ PCDFs and the different classes of PCBs typically investigated in environmental studies. An additional merit of this HPLC stationary phase is possible automation. Table 3.2 shows some examples in which different adsorbents were used in the fractionation step of cleanup methodology. It is worth noting that immunoaffinity chromatography (IAC) using monoclonal and polyclonal antibodies specifically developed to recognize 2,3,7,8-PCDD/PCDFs was considered a very attractive technique in the 1990s. Because of the good results achieved in the cleanup of aqueous samples (water and blood) for dioxin analysis (Concejero et al. 2004), it was initially thought to be very promising. However, the need for using fatfree extracts, the variability of the results, and the presence of cross-reactions rendered this alternative cleanup process without further applications.

Automation of extraction and cleanup In view of the extreme difficulty and tediousness of the extraction and cleanup process in food dioxin analysis, there have been many attempts at automation, but so far no one has come up with an automated procedure for simultaneous extraction and cleanup. Smith and coworkers (1984) made the first attempt at a semiautomated at-line extraction/ cleanup procedure. They developed a method for dioxin analysis of biological tissues (including fish) in two steps. In the first step, the extraction and a first cleanup step using active carbon were performed simultaneously. In the second step, the extract was applied to a second series of adsorbents

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

contained in two tandem columns. Based on this general scheme, in 1997 a semiautomated method for online extraction plus cleanup and fractionation of PCBs and PCDD/PCDFs was developed (Krokos et al. 1997). Up to 6 g of fat could be extracted with this method, which is very useful in the case of fatty food samples, as demonstrated by several studies (Jiménez et al. 1996; Serrano et al. 2000; Santillo et al. 2003). Nevertheless, despite its good performance, the method has not been widely used, as no apparatus is available commercially. The efficiency of the automated PowerPrep system in purifying sample extracts for dioxin analyses has already been demonstrated in recent years for different types of matrices, including food (Eljarrat 2001; Pirard et al. 2002). The multistep procedure is based on the use of disposable multilayer silica columns, basic alumina, and PX-21 carbon columns, which can be combined to suit the target analytes. This means that dioxins and non-ortho PCBs can be isolated in a fraction with good recoveries, and several samples can be analyzed in parallel, even those with high fat contents (Focant et al. 2001). Table 3.2 shows that the Power-Prep system is today used as a routine cleanup method for dioxin food analysis. This method has become increasingly popular over the years. As the only commercially available apparatus, it is gradually making its way into all laboratories that perform large numbers of analyses. Some efforts have been made to couple an online PLE (or ASE) extraction step, but for the moment, no satisfactory results have been achieved (Focant et al. 2004). Although laboratories can choose the method that best suits them for extraction plus cleanup of dioxins in foodstuffs, there is no doubt that PLE (or ASE) as an extraction method and the Power-Prep system for cleanup, both commercial products, are the only methods that, although not fully automated, permit large numbers of samples to be

61

analyzed in the shortest possible time. With this combination, it is possible to handle 10 samples at once in both the extraction and the purification steps and to deal with any food health emergency due to dioxin contamination of foodstuffs.

Instrumental determination As noted above, the choice of analytical procedure for extraction plus cleanup of the seventeen 2,3,7,8-PCDD/PCDF congeners is up to each laboratory, provided that the analyte recoveries are within the range laid down by the EU directive (Commission Regulation 1883/2006/EC). However, in the case of the instrumental determination, the EU directive requires the use of GC-HRMS, which was the only method able to reliably determine dioxins at levels appropriate for food analysis. Some other methods, such as DR CALUX bioassay and GC coupled to ion trap MS working in tandem mode, or GC-MS/MS(ITD), are accepted officially only for screening purposes. The most important specific requirements are recovery control by the addition of 13C12PCDD/PCDFs as standard. The recoveries of the individual internal standards should be between 50% and 130%. The GC separation of the isomers should be <25% peak to peak, and the identification should be performed according to EPA Method 1613 revision B and the EU official method (Commission Regulation 1883/2006/EC) using isotope dilution HRGC/HRMS. The difference between the upper bound (not detected at limit of detection) and lower bound (not detected equal to zero) determination levels should not exceed 20% for foodstuffs with about 1 pg WHO-TEQ/g fat (only PCDD/ PCDF) and 25–40% for foodstuffs with about 0.5 pg WHO-TEQ/g fat. The assessment of all is mandatory in order to meet the basic requirements of high sensitivity, selectivity, specificity, accuracy, and precision of the analytical procedure.

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Analysis of Endocrine Disrupting Compounds in Food

Following a number of dioxin contamination incidents involving foodstuffs (Malisch 2000; Bernard et al. 2002, Hoogenboom et al. 2007), there has been a tremendous increase in the demand for fast and low-cost PCDD/ PCDF measurements in food. Because of this demand, alternative and relatively inexpensive techniques, such as GC-MS/MS (ITD) (Malavia et al. 2007a, b) and comprehensive two-dimensional GC (GCxGC) with electron capture detection (GCxGC-μECD) (Danielsson et al. 2005) or MS detection (GCxGC-ToF-MS) (Focant et al. 2005b), have recently been developed and validated in order to be accepted as alternative confirmation methods to GC-HRMS for the instrumental determination of PCDD/PCDFs.

GC congener separation High-resolution GC methods for the analysis of PCDD/PCDFs have been developed extensively in the last two decades and continue to progress today. Ryan and coworkers reported in 1991 the GC isomer-specific separation of all 136 tetra- to octa-PCDD/PCDFs on a series of nine fused-silica capillary GC columns containing silicone stationary phases of diverse polarity (100% methyl; 5% phenyl methyl; 50% phenyl methyl; 50% methyl trifluoropropyl; 50%, 75%, 90%, and 100% cyanopropyl; and liquid crystalline smectic) (Ryan et al. 1991). The authors showed that all 136 PCDD/PCDF compounds could be separated from each other by a combination of a minimum of two stationary phases. More recent studies have focused on the separation of the seventeen 2,3,7,8PCDD/PCDF congeners that have assigned a toxic equivalency factor (TEF) from closely coeluting isomers. Almost all methods found in the literature for dioxin analysis in food samples use 5% diphenyl–95% dimethyl polysiloxane type stationary phases, such as DB-5 (J&W Scientific, Folsom, CA, USA) or equivalent (Choi et al. 2002; Abad et al. 2002; Kiviranta et al. 2004; Bordajandi et al. 2004).

However, because the introduction in the market of 5% silphenylene silicone copolymer or Si-arylene type stationary phase, that is, DB-5MS (J&W Scientific), most laboratories use this column or the equivalent for dioxin analysis. Besides an increased thermal stability (Abad et al. 1997), DB-5MS provides a somewhat different selectivity toward certain isomers as compared to DB-5. However, neither of these two GC stationary phases can completely separate all seventeen 2,3,7,8-PCDD/PCDF congeners, particularly the 2,3,7,8-TCDF. The use of dioxin-selective stationary phases, such as Rtx-Dioxin 2 (Restek, Bellefonte, PA, USA), has not provided complete separation among target coeluting isomers (i.e., 1,2,3,7,8- and 1,2,3,6,7-penta-chlorinated dioxins) (Cochran et al. 2007). In a recent comprehensive study, a total of 13 different GC columns were evaluated, including not only 5% diphenyl siloxane stationary phases from different manufacturers but also low- and high-polarity GC columns for the separation of the 17 target dioxin congeners: HP-5MS (Agilent Technologies, Santa Clara, CA, USA); Rtx-5MS and Rtx-Dioxin-2 (Restek Corp.); Supelco Equity 5 and SP-2331 (Supelco, Bellefonte, PA, USA); Factor Four VF-5MS and CP-Sil 8 CB LowBleed/MS (Varian, Walnut Creek, CA, USA); DB-5, DB-5MS, DB-225, DB-XLB (J&W Scientific); and ZB-5MS and ZB-5UMS (Phenomenex Inc., Torrance, CA, USA) (Fishman et al. 2007). Their conclusion was similar to that of Ryan and coworkers (1991): all dioxins can be separated from closely eluting isomers using two sets of nonpolar and polar stationaryphase combinations. In fact, EPA Method 1643 and the European Standard Method recommend the use of a primary GC column followed by a confirmation analysis using a polar GC stationary phase, such as DB-255 (50% cyanopropylmethyl, 50% phenylmethylsiloxane); DB-Dioxin (44% methyl, 28% phenyl, 20% cyanopropyl polixiloxane) (J&W Scientific); or Supelco SP-2330 (100%

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

cyanopropyl polysiloxane) or equivalent, as a complementary tool (U.S. EPA 2002; Commission Regulation 1883/2006/EC). In a recent interlaboratory comparison of the determination of 2,3,7,8-PCDD/PCDFs, the authors recommend two different primary and confirmation column systems for accurate quantification, that is, DB-5/SP-225 or, alternatively, DB-5MS/SP-2331 (Wilken et al. 2008). Besides the efforts to develop columns to separate all 2,3,7,8-substituted congeners and systems using dual GC ovens to accommodate the use of two GC columns and improve the efficiency and speed of the analysis (Fishman et al. 2008), multidimensional GC techniques, such as heart-cut multidimensional gas chromatography (heart-cut MDGC) and especially GCxGC, are also regarded as powerful alternatives to the one-dimensional GC to solve coelutions between dioxins and other similar compounds present in the extract. Heart-cut MDGC allows coeluting congeners on a precolumn to be transferred to a second capillary column with a different selectivity, improving the separation of the selected regions. However, when dealing with such complex mixtures, the number of heart-cuts that can be made in one analytical run is limited, making it necessary to reinject the extract several times to avoid coelutions in the second column. This fact makes this technique time consuming, and thus the number of applications for PCDD/PCDF analysis in the literature is scarce (Schomburg et al. 1985; Jia et al. 1991). Recently, GCxGC has been recognized as a powerful chromatographic technique for the resolution of complex mixtures. In this case, a modulation process transfers the entire effluent from the first column into the second as consecutive narrow bands. Compared to heart-cut MDGC, a much higher peak capacity is obtained because the whole extract is subjected to two independent chromatographic separations by two sequential GC columns, without the need to re-inject the same extract several

63

times. In addition, the focusing effect that takes place during the modulation yields an increase in the signal-to-noise ratio, improving the limits of detection (Dallüge et al. 2003). Since its introduction at the beginning of the 1990s by Liu and Phillips (1991), the number of applications of GCxGC in the environmental and food fields has grown exponentially, thanks to the advances in the instrument setup, such as more robust interfaces (modulators) between the first and second capillary columns and the possibility of coupling to a number of detection systems. The combination of GCxGC with microelectron detection (GCxGC-μECD) has been regarded as a promising technique for the determination of PCDD/PCDF (Haglund et al. 2008). Besides providing sufficient selectivity and sensitivity for their reliable determination at low levels in complex matrices, it would be a more cost-efficient option than GC-HRMS, the official confirmatory method for the control of dioxins in food (Commission Regulation 1883/2006/EC). The selection of the stationary phase of the first- and seconddimension columns is again a critical step. A number of column combinations have been evaluated for the complete separation of the 17 priority PCDD/PCDF isomers and the 12 WHO-PCBs (that have an assigned TEF value) from each other and from other compounds that could be present in the extracts. Korytár et al. (2004) found that the combination of a nonpolar stationary phase, such as DB-XLB, in the first dimension, and the liquid-crystalline LC-50 (J&K Environmental, Milton, ONT, Canada), as the second dimension, provided the complete separation of the 29 priority congeners from each other as well as from matrix constituents. In a further study, Danielsson et al. (2005) used the same column combination (DB-XLB × LC-50) for the analysis of food samples. The TEQ data compared well to the results obtained by GC-HRMS, indicating that although a more intensive validation should be performed in order to propose

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Analysis of Endocrine Disrupting Compounds in Food

GCxGC-μECD as a complementary/ confirmatory method, it has great potential as a screening method because it provides not only the TEQs value but also the congener distribution profile in the samples (Haglund et al. 2008). Certainly, the coupling of GCxGC with MS has additional advantages to μECD, including the possibility of using isotope dilution for quantification, but at increasing costs (Mondello and Tranchida 2008). Up to now, most of the studies have been carried out using ToF-MS. Focant et al. (2005b) explored the possibilities of GCxGC-ToFMS with the column combination Rtx500 × BPX-50. The TEQ results obtained compared favorably to those obtained by GC-HRMS for fish samples, and for samples with lower PCDD/PCDF concentrations, such as pork and milk, overestimation for some congeners was observed as was a higher coefficient of variation (CV) compared to GC-HRMS. Hoh and coworkers (2007, 2008) also explored the possibilities of GCxGCToF-MS for the analysis of the 17 target PCDD/PCDFs. The column combination Rtx-Dioxin 2 × Rtx-PCB allowed the determination of the target analytes without interference by using deconvolution software. However, when the method was applied to fish oil samples, the limits of quantification were too high to fulfill the requirements, as in the current regulatory levels for congenerspecific dioxin analysis (Hoh et al. 2008). On the other hand, the introduction to the market of rapid-scanning quadrupole MS instruments with a lower cost than ToF-MS systems is promising, making it possible to use electron-capture negative ionization (ECNI) instead of electronic impact (EI) to enhance, in many cases, the detectability of the analyte (Korytár et al. 2005). Improvements in the general setup of the GCxGC system and advances in the MS detectors will enable this technique to become fully established in routine analysis laboratories for dioxin analysis, provided that more user-friendly software

for instrument control, visualization, and data treatment are available.

GC detectors According to the European regulations, and taking into account the very low PCDD/ PCDF concentration levels found in almost all types of food samples, HRMS is the most commonly used detection system. However, due to its high cost and maintenance, over the last few years some authors have focused their research on the quest for suitable alternatives to this technique. One of the most promising alternatives is based on tandem in-time MS using ion trap detectors (ITDs). Theoretically, this operation mode is able to decrease the background noise considerably, increasing the signal-to-noise ratios and thus providing lower limits of detection (LODs). The other promising alternative is the use of ToF-MS detectors. Although these MS instruments are not known for their great sensitivity, their fast acquisition rates makes it possible to couple ToF-MS with GCxGC. As was previously mentioned, the modulation process associated with the GCxGC separation technique improves the signal-to-noise ratio, leading to a decrease of the LODs obtained, even when a ToF detector is used. However, applications of both techniques to dioxin determination in food are still scarce, and the use of GCxGC-ToF-MS is neither cheaper nor easier than HRMS. Table 3.4 summarizes the LODs reported for the determination of PCDD/PCDF in several foodstuffs from different countries and in different periods of time using three different detection systems: HRMS, MS/MS (ITD), and ToF-MS (in this last case, coupled to GCxGC). It is difficult to compare the LODs obtained using the different MS systems because these values are not only dependent on instrumental performance but also on the sample treatment procedures employed and the type of sample analyzed (as can be observed, for example, in the case of

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

65

Table 3.4. LODs of different detection systems used for PCDD/PCDF determinations in food samples collected in different countries and years. Detection System HRMS

ITD (MS/MS)

(GC×GC)ToF

Type of Fooda

Country

Sampling Period

LOD

References

a–h

Spain

2002

0.02–0.20 pg/g (d.w.)

Bocio and Domingo 2005 Fernández et al. 2004 Gómara et al. 2005

a–d, h

Spain

2000–2003

0.001–2.19 pg/g (l.w.)

a

Spain

1995–2003

c (breast milk) b

Belgium

2000–2001

0.00001–0.34 pg/g (f.w.) 0.1–1.9 pg/g (l.w.)

Belgium

1999

b–c g a f a–d, f–h b

Finland Finland Finland Finland Finland USA

1998–2000 1998–2000 1998–2000 1998–2000 1997–1999 1997

<0.2 pg/g (tetra- to hexa-PCDD/PCDFs) (n.i.) 0.1–1.0 pg/g (n.i.) 0.005–0.05 pg/g (n.i.) 0.01–0.1 pg/g (n.i.) 0.5–5 pg/g (n.i.) 0.0007–0.63 pg/g (f.w.) 0.05–1.00 pg/g (l.w.)

a–b a–c, f–h a–h Drinking water Tea a

India Taiwan Japan Japan

2000 1993–1996 1994–2001 1994–2001

0.1–10 pg/g (l.w.) 0.042–0.922 pg/g (f.w.) 0.01–0.02 pg/g (n.i.) 0.0001–0.0002 pg/L

Kiviranta et al. 2001 Kiviranta et al. 2001 Kiviranta et al. 2001 Kiviranta et al. 2001 Kiviranta et al. 2004 Ferrario and Byrne 2000 Kumar et al. 2001 Wang et al. 2009 Sasamoto et al. 2006 Sasamoto et al. 2006

Japan Australia

2006 2005–2006

0.01–0.05 pg/g (n.i.) 0.1–76 pg/g (l.w.)

Amakura et al. 2009 Matthews et al. 2008

b, c, f

RM/IE

RM/IE

Eppe et al. 2004

a–d d b–c e Infant formulas a, c, f f

RM/IE RM/IE RM/IE RM/IE RM/IE

RM/IE RM/IE RM/IE RM/IE RM/IE

200 fg/μL injected (TCDD) 0.09–0.36 pg injected 0.04–0.20 pg/g (l.w.) 0.10–0.93 pg/g (l.w.) 0.06–0.35 pg/g (l.w.) 0.1–8.7 pg/g (l.w.)

USA USA

1998–1999 RM/IE

0.03–0.1 pg/g (n.i.) 0.01–0.03 pg/g (TCDD) (n.i.)

Hayward et al. 2001 Hayward et al. 1999

—

RM/IE

RM/IE

Hoh et al. 2007

e

RM/IE

RM/IE

a–c

RM/IE

RM/IE

0.25 pg injected (TCDD) 0.019–7.8 pg/g (TEQ) (n.i.) 0.2–0.5 pg injected

Focant et al. 2002b Covaci et al. 2002

Malavia et al. 2008 Malavia et al. 2007a Malavia et al. 2007b Malavia et al. 2007b Loran et al. 2007

Hoh et al. 2008 Focant et al. 2005b

a

(a) fish and sea fish, (b) meat and meat products, (c) dairy products, (d) vegetable oil and butter, (e) fish oil, (f) eggs, (g) vegetables and fruits, (h) cereals and pulses. d.w., dry weight; f.w., fresh weight; l.w., lipid weight; n.i., not indicated; RM/IE, reference matrix or interlaboratory exercise.

drinking water). Nevertheless, some general observations can be made from the data gathered in Table 3.4. The number of applications in which MS alternatives to HRMS are used are very scarce in the literature. In almost half of these cases, the LODs are calculated using

standard solutions (Eppe et al. 2004; Malavia et al. 2008; Hoh et al. 2007; Focant et al. 2005b,c) or calculated only for the most sensitive congener, the TCDD (Hayward et al. 1999). In addition, the applications of these detection systems are usually limited to

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Analysis of Endocrine Disrupting Compounds in Food

the determination of dioxins in foodstuffs with a high lipid content and thus with high dioxin concentration levels, such as fish oils (Hoh et al. 2008; Malavia et al. 2007a,b). Today, there are in the market several MS systems able to perform MS/MS experiments (triple-quadrupole) and others able to achieve resolution values close to those typical of the instruments based on magnetic sector technology (the most common for dioxin analyses), such as hybrid instruments (e.g., QqToF and LTQ Orbitrap). However, the high acquisition cost does not make these instruments a low-cost alternative to (magnetic sector) HRMS, being applied only for a few instrumental-method development proposes and not for routine analysis of PCDD/ PCDF in the laboratories.

Bioanalytical screening methods Several dioxin food incidents (Malisch 2000; Bernard et al. 2002; Hoogenboom et al. 2007) and also recent EU regulations (Commission Regulation 1883/2006/EC) highlight the need for screening methods for food and feed analysis. This need is even more acute during a contamination incident, first to rapidly locate the source and second to reserve the oftenlimited GC-HRMS capacity for the confirmation analysis of suspected samples. Several bioanalytical detection methods (BDMs) for measuring dioxin-like activity have been developed since the early 1990s. These methods are based on the ability of key biological molecules to recognize a unique structural property of dioxins or to respond to dioxins in a specific way. Most bioassays are based on the assumption that dioxin compounds act through the aryl hydrocarbon receptor (AhR) signal transduction pathway. The biological methods include biomarkers (e.g., wildlife/human effects) (Schecter 1994); whole animal exposures (in vivo, laboratory exposure) (van den Berg et al. 2006); cell-based or organ-based bioassays (e.g., EROD, in vitro luciferase) (Jones et al. 2000);

and protein-binding assays (e.g., ligand binding and immunoassays) (Díaz-Ferrero et al. 1997; Seidel et al. 2005). The good results obtained with an enzyme-linked immunoassay (ELISA) using monoclonal antibodies for dioxin-like PCBs in food samples (Tsutsumi et al. 2008) makes the use of bioassays promising for fast and reliable dioxin food analysis. From the methods mentioned above, the one that has achieved the most popularity and that is accepted by the new EU regulations as a screening method is the DR CALUX bioassay. This method uses genetically modified rat or mouse hepatoma cells that respond to chemicals that activate the AhR. The recombinant CALUX cells contain a stably transfected AhR-responsive firefly luciferase reporter gene, which responds to dioxins and dioxin-like chemicals. At present, the different cell lines are commercialized and sold as the DR CALUX assay; one is based on modified rat H4IIE hepatoma cells (GudLuc1.1), and the other is based on modified H1L6.1 mouse hepatoma cells. The rat cells appear to be more sensitive, showing response at TCDD concentrations below 1 pM (Goeyens et al. 2004). Recently, there have been a number of international validation studies, such as ring trials on different foodstuffs under the EUsponsored DIFFERENCE project (van Loco et al. 2004). The results for both methodologies were comparable and within the consensus values. Bovee and coworkers (1998) were the first to show their utility for screening milk fat near the existing limit of 6 pg TEQ/fat. Based on this work, the test was validated for other food matrices (van Leeuwen et al. 2007; Hasegawa et al. 2007). However, all stressed that exhaustive cleanup (similar to that necessary for GC-HRMS) is essential to ensure accurate results, which means that one of the advantages of using biological analysis, rapidity, is lost. Therefore, there are some issues that still need improvement, mainly relating to the cleanup procedure. In this way, there have

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

been some recent papers dealing with the identification of natural aryl-receptor agonists present in foodstuffs that are detected with DR CALUX bioassay (van Ede et al. 2008).

Quality assurance and quality control Performance criteria for GC-HRMS isomerspecific analysis have been given previously (U.S. EPA 2002; Commission Regulation 1883/2006/EC). The quality of the analysis should include blanks, duplicate samples, and recovery experiments in each series of samples. Furthermore, certified reference materials (CRMs) should be analyzed frequently. In addition, periodic interlaboratory studies, such as the those organized by the QUASIMEME project or the Norwegian Institute of Public Health (Folkehelseinstituttet), and proficiency testing schemes must be carried out periodically to check data comparability. However, most reports on PCDD/PCDF concentrations in food do not describe validation data, and only a few provide details on the quality control procedure followed. Quality assurance/quality control (QA/QC) plans are essential elements for any laboratory and any analytical procedure in order to comply with the EU and EPA criteria for the determination of PCDD/PCDF in food and food products. In addition, accreditation for the ISO/IEC 17025 standard is mandatory for food control laboratories.

References Abad, Esteban; Caixach, Josep; Rivera, Josep. 1997. Application of DB-5ms gas chromatography column for the complete assignment of 2,3,7,8-substituted polychlorodibenzo-p-dioxins and polychlorodibenzofurans in samples from municipal waste incinerator emissions. J. Chromatogr. A. 786(1):125–134. Abad, Esteban; Llerena, Juliá J.; Sauló, Jordi; Caixach, Josep; Rivera, Josep. 2002. Study on PCDDs/PCDFs and co-PCBs content in food samples from Catalonia (Spain). Chemosphere. 46:1435–1441. Ahmed, Farid E. 2001. Analysis of pesticides and their metabolites in foods and drinks. Trends Anal. Chem. 20:649–661.

67

Ahmed, Farid E. 2003. Analysis of polychlorinated biphenyls in food products. Trends Anal. Chem. 22:170–185. Ahn, Yun Gyong; Seo, Jungju; Shin, Jeoung Hwa; Khim, Jeehyeong; Hong, Jongki. 2006. Development of new cleanup method of polychlorinated dibenzo-p-dioxins/ dibenzofurans in fish by freezing-lipid filtration. Anal. Chim. Acta. 576:31–36. Amakura, Yoshiaki; Tsutsumi, Tomoaki; Tanno, Kenji; Nomura, Koichi; Yanagi, Toshihiko; Kono, Yoichi; Yoshimura, Morio; Maitani, Tamio; Matsuda, Rieko; Yoshida, Takashi. 2009. Dioxin concentrations in commercial health tea materials in Japan. J. Health Sci. 55:290–293. Baars, J. Albert; Bakker, Martin I.; Baumann, Robert A.; Boon, Polly E.; Freijer, Jan I.; Hoogenboom, Laurentius A.P.; Hoogerbrugge, Ronald; van Klaveren, Jacob; Liem, A.K. Djien; Traag, Wim A.; de Vries, Jan. 2004. Dioxins, dioxin-like PCBs, and non-dioxin-like PCBs in foodstuffs: Occurrence and dietary intake in The Netherlands. Toxicol. Lett. 151:51–61. Bernard, Alfred; Broeckaert, Fabrice; De Poorter, Geert; De Cock, Ann; Hermans Cédric; Saegerman, Claude; Houins, Gilbert. 2002. The Belgian PCB/dioxin crisis: Analysis of the food chain contamination and health risk evaluation. Environ. Res. 88:1–18. Bernsmann, Thorsten; Fürst, Peter. 2004. Comparison of accelerated solvent extraction (ASE) with integrated sulphuric acid clean up and Soxhlet extraction for determination of PCDD/PCDF, dioxin-like PCB, and indicator PCB in feeding stuffs. Organohalogen Compd. 66:159–163. Björklund, Erland; Müller, Anne; von Holst, Christoph. 2001. Comparison of fat retainers in accelerated solvent extraction for the selective extraction of PCBs from fat-containing samples. Anal. Chem. 73:4050– 4053. Björklund, Erland; Sporring, Sune; Wiberg, Karin; Haglund, Peter; von Holst, Christoph. 2006. New strategies for extraction and clean-up of persistent organic pollutants from food and feed samples using selective pressurised liquid extraction. Trends Anal. Chem. 25:318–325. Bocio, Ana; Domingo, Jose L. 2005. Daily intake of polychlorinated dibenzo-p-dioxins /polychlorinated dibenzofurans (PCDD/Fs) in foodstuffs consumed in Tarragona, Spain: A review of recent studies (2001– 2003) on human PCDD/F exposure through the diet. Environ. Research. 97:1–9. Bordajandi, Luisa R.; Gómez, Gema; Abad, Esteban; Rivera, Josep; Fernández-Bastón, M. Mar; Blasco, Julián; González, María José. 2004. Survey of persistent organic pollutants (PCBs, PCDD/Fs, PAHs), heavy metals (Cu, Cd, Zn, Pb, Hg), and arsenic in food samples from Huelva (Spain): Levels, congener distribution, and health implications. J. Agric. Food Chem. 52:992–1001. Bordajandi, Luisa R.; Gómara, Belén; González, Maria José. Analysis of dioxins in seafood and seafood products. 2010. Handbook of Seafood and Seafood Products Analysis. L. Nollet and F. Toldra, editors.

68

Analysis of Endocrine Disrupting Compounds in Food

CRC Press, Boca Raton, Florida, USA. Chapter 40, pp. 797–815. Bovee, Toine F.H.; Hoogenboom, Laurentius A.P.; Hamers, Astrid R.M.; Traag, Wim A.; Zuidema, Tina; Aarts, Jac M.M.J.G.; Brouwer, Abraham; Kuiper, Harry A. 1998. Validation and use of the CALUXbioassay for the determination of dioxins and PCBs in bovine milk. Food Addit. Contam. 15:863–875. Camel, Valérie. 2000. Microwave-assisted solvent extraction of environmental samples. Trends Anal. Chem. 19:229–248. Chang, Ruth R.; Jarman, Walter M.; Hennings, John A. 1993. Sample cleanup by solid phase extraction for the ultratrace determination of polychlorinated dibenzo-pdioxins and dibenzofurans in biological samples. Anal. Chem. 65:2420–2427. Chen, Hsiu-Ling; Su, Huei-Jen; Hsu, Jing-Fang; Liao, Pao-Chi; Lee, Ching-Chang. 2008. High variation of PCDDs, PCDFs, and dioxin-like PCBs ratio in cooked food from the first total diet survey in Taiwan. Chemosphere. 70:673–681. Choi, Dongmi; Hu, Soojung; Jeong, Jiyoon; Won, Kyungpoong; Song, Insang. 2002. Determining dioxin-like compounds in selected Korean food. Chemosphere. 46:1423–1427. Cochran, Jack; Dorman, Frank; Stidsen, Gary; Reese, Shawn; MacPherson, Karen; Kolic, T; Reiner, Eric; Ryan, John J.; Bradley, Joe; Craig, D.; Priest, Benjamin. 2007. Retention time profiling for all 136 tetra- through octa-chlorinated dioxins and furans on a unique, low-bleed, thermally-stable gas chromatography column. Organohalogen Compd. 69:477– 480. Commission Regulation (EC) No 76/2000/EC setting maximum levels for dioxins in emissions of municipal waste incinerators. Official Journal of the European Communities L 321/91–100, 2000. Commission Recommendation (EC) No 201/2002/EC on the reduction of dioxins, furans and PCBs in feedingstuffs and foodstuffs. Official Journal of the European Communities L836/67–73, 2002. Commission Regulation (EC) No 1883/2006/EC of 19 December 2006 laying down methods of sampling and analysis for the official control of levels of dioxins and dioxin-like PCBs in certain foodstuffs. Official Journal of the European Communities L 364, 20.12.2006, p 32–43. Council Regulation (EC) No 1881/2006/EC amending Regulation (EC) No 466/2001 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Communities L 364, 20.12.2006, p 5–24. Concejero, Miguel. A.; Ramos, Lourdes; Jiménez, Begoña; Gómara, Belén; Abad, Esteban; Rivera, Josep; González, María José. 2001. Suitability of several carbon sorbents for the fractionation of various sub-groups of toxic polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans. J. Chromatogr. A. 917:227–237. Concejero, Miguel A.; Abad, Esteban; Rivera, Josep; Herradón, Bernardo; González, María José; de Frutos, Mercedes. 2004. Different retention of dioxin-like compounds and organochlorinated insecticides on an

immunochromatographic column. Interpretation and applicability. J. Sep. Sci. 27:1093–1101. Covaci, Adrian; Ryan, John Jake; Schepens, Paul. 2002. Patterns of PCBs and PCDD/PCDFs in chicken and pork fat following a Belgian food contamination incident. Chemosphere. 47:207–217. Creaser, Colin. S; Al-Haddad, Ameera. 1989. Fractionation of polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans on porous graphitic carbon. Anal. Chem. 61:1300–1302. Dallüge, Jens; Beens, Jan; Brinkman, Udo A.Th. 2003. Comprehensive two-dimensional gas chromatography: A powerful and versatile analytical tool. J. Chromatogr. A. 1000:69–108. Danielsson, Conny; Wiberg, Karin; Korytár, Peter; Bergek, Sture; Brinkman, Udo A. Th.; Haglund, Peter. 2005. Trace analysis of polychlorinated dibenzo-pdioxins, dibenzofurans and WHO polychlorinated biphenyls in food using comprehensive twodimensional gas chromatography with electroncapture detection. J. Chromatogr. A. 1086:61–70. de Boer, Jacob. 1988. Trends in chlorobiphenyl contents in livers of Atlantic cod (Gadus morhua) from the North Sea, 1979–1987. Chemosphere. 17:1811– 1819. de Boer, Jacob; Stronk, C.J.N.; Traag, Win A.; van der Meer, J. 1993. Non-ortho and mono-ortho substituted chlorobiphenyls and chlorinated dibenzo-p-dioxins and dibenzofurans in marine freshwater fish and shellfish from the Netherlands. Chemosphere. 26:1823– 1842. de Boer, Jacob; Lau, Robin J. 2003. Developments in the use of chromatographic techniques in marine laboratories for the determination of halogenated contaminants and polycyclic aromatic hydrocarbons, J. Chromatogr. A. 1000:223–251. Díaz-Ferrero, Jordi; Rodríguez-Larena, M. Carmen; Comellas, Luis; Jiménez, Begoña. 1997. Bioanalytical methods applied to endocrine disrupting polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans. A review. Trends Anal. Chem. 16:563–573. Díaz-Ferrero, Jordi; Grau, Jordi; Montana, M. Jesús; Martí, Ramón. 2005. Study of dioxins, furans, and polychlorinated biphenyl fractionation on HPLC using a pyrenil column for their analysis in meat and fish samples. Afinidad. 62:433–438. Dionex Application Note 322. 1996. Dionex, Sunnyvale, CA, USA. Available at: http://www.dionex.com/ en-us/webdocs/4324_AN322.pdf El-Kady, Ahmend A.; Abdel-Wahhab, Mosaad A.; Henkelmann, Bernhard; Belal, Mohamed H.; Morsi, M. Khairy S.; Galal, Samy M.; Schramm, KarlWerner. 2007. Polychlorinated biphenyl, polychlorinated dibenzo-p-dioxin, and polychlorinated dibenzofurans residues in sediments and fish of the River Nile in the Cairo region. Chemosphere. 68: 1660–1668. Eljarrat, Ethel; Caixach, Josep; Rivera, Josep. 1998. Microwave vs. Soxhlet for the extraction of PCDDs and PCDFs from sewage sludge samples. Chemosphere. 36:2359–2336.

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

Eljarrat, Ethel. 2001. Evaluation of an automated cleanup system for the isotope-dilution high resolution mass spectrometry analysis of PCB, PCDD, and PCDF in food. Fresenius J. Anal. Chem. 371:983– 988. Eppe, Gauthier; Focant, Jean-François; Pirard, Catherine; de Pauw, Edwin. 2004. PTV-LV-GC/MS/MS as screening and complementary method to HRMS for the monitoring of dioxin levels in food and feed. Talanta. 63:1135–1146. Fernandes, Alwyn; Shaun, White; Kyle, D’Silva; Martin, Rose. 2004 Simultaneous determination of PCDDs, PCDFs, PCBs, and PBDEs in food. Talanta. 63: 1147–1155. Fernández, Mario A.; Gómara, Belén; Bordajandi, Luisa R.; Herrero, Laura; Abad, Esteban; Ábalos, Manuela; Rivera, Josep; González, María José. 2004. Dietary intakes of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in Spain. Food Add. Contam. 21:983–991. Ferrario, Joseph; Byrne, Christian. 2000. The concentration and distribution of 2,3,7,8-dibenzo-pdioxins/-furans in chickens. Chemosphere. 40:221– 224. Fiedler, Heidelore. 2007. National PCDD/PCDF release inventories under the Stockholm Convention on persistent organic pollutants. Chemosphere. 67:S96– S108. Fishman, Vyacheslav N.; Martin, Gregory D.; Lamparski, Lester L. 2007. Comparison of a variety of gas chromatographic columns with different polarities for the separation of chlorinated dibenzo-p-dioxins and dibenzofurans by high-resolution mass spectrometry. J. Chromatogr. A. 1139:285–300. Fishman, Vyacheslav N.; Martin, Gregory D.; Wilken Michael. 2008. Discussion on the separation of 2378-substituted isomers from al 136 tetra- through octa-polychlorinated dibenzo-p-dioxins and dibenzofurans on Si-arylene stationary phase. Organohalogen Compd. 70:473–476. Focant, Jean-François; Eppe, Gauthier; Pirard, Catherine; De Pauw, Edwin. 2001. Fast clean-up for polychlorinated dibenzo-p-dioxins, dibenzofurans, and coplanar polychlorinated biphenyls analysis of high-fatcontent biological samples. J. Chromatogr. A. 925: 207–221. Focant, Jean-François; Eppe, Gauthier; Pirard, Catherine; Massart, Anne-Cécile; André, J. E; De Pauw, Edwin. 2002a. Levels and congener distribution of PCDDs, PCDFs, and non-ortho PCBs in Belgian foodstuff. Assessment of dietary intake. Chemosphere. 48:167– 179. Focant, Jean-François; Pirard, Catherine; Thielen, Caroline; De Pauw, Edwin. 2002b. Levels and profiles of PCDDs, PCDFs, and cPCBs in Belgian breast milk. Estimation of infant intake. Chemosphere. 48:763– 770. Focant, Jean-François; Pirard, Catherine; Massart, AnneCécile; De Pauw, Edwin. 2003. Survey of commercial pasteurised cows’ milk in Wallonia (Belgium) for the occurrence of polychlorinated dibenzo-p-dioxins, dibenzofurans, and coplanar polychlorinated biphenyls. Chemosphere. 52:725–733.

69

Focant, Jean-François; Pirard, Catherine; De Pauw, Edwin. 2004. Automated sample preparationfractionation for the measurement of dioxins and related compounds in biological matrices: A review. Talanta. 63:1101–1113. Focant, Jean-François; Pirard, Catherine; Massart, AnneCécile; Scholl, Georges; Eppe, Gauthier; De Pauw, Edwin. 2005a. Integrated PLE multi-step automated clean up and fractionation for the measurement of dioxins and PCBs in food and feed. Organohalogen Compd. 67:261–264. Focant, Jean-François; Eppe, Gauthier; Scippo, MarieLouise; Massart, Anne-Cécile; Pirard, Catherine; Maghuin-Rogister, Guy; De Pauw, Edwin. 2005b. Comprehensive two-dimensional gas chromatography with isotope dilution time-of-flight mass spectrometry for the measurement of dioxins and polychlorinated biphenyls in foodstuffs: Comparison with other methods. J. Chromatogr. A. 1086:45–60. Focant, Jean-François; Pirard, Catherine; Eppe, Gauthier; De Pauw, Edwin. 2005c. Recent advances in mass spectrometric measurement of dioxins. J. Chromatography A. 1067:265–275. Goeyens, Leo; Windal, Isabelle; Scippo, Marie-Louise; Nathalue, Van Wouwe; Eppe, Gauthier; De Pauw, Edwin; Maghin-Rogister, Guy. 2004. Comparison of the rat and mouse cell lines commercially available for CALUX bioassays. Organohalogen Compd. 66:608– 615. Gómara, Belén; Bordajandi, Luisa R.; Fernández, Mario A.; Herrero, Laura; Abad, Esteban; Ábalos, Manuela; Rivera, Josep; González, Maria José. 2005. Levels and trends of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (PCBs) in Spanish commercial fish and shellfish products, 1995–2003. J. Agric. Food Chem. 53:8406– 8413. Gómara, Belén; Herrero, Laura; González, María José. 2006. Survey of polybrominated diphenyl ether levels in Spanish commercial foodstuffs. Environ. Sci. Technol. 40:7541–7547. Grochowalski, Adam Jan; Wojtalewicz, Dominika. 2005. Food samples clean-up using semipermeable polyethylene membranes (SPM) for the determination of polybrominated diphenyl ethers. Organohalogen Compd. 67:258–260. Grümping, Rainer; Malte, Petersen; Frank, Neugebauer; Matthias, Opel. 2008. Levels of dioxins, PCBs, BFRs, PFCs, and organotins in fishery products from Latvia. Organohalogen Compd. 70:582–585. Haglund, Peter; Korytár, Peter; Danielsson, Conny; Díaz, Jordi; Wiberg, Karin; Leonards, Pim; Brinkman, Udo A. T.; de Boer, Jacob. 2008. GC × GC-ECD: A promising method for the determination of dioxins and dioxin-like PCBs in food and feed. Anal. Bioanal. Chem. 390:1815–1827. Hasegawa, Jun; Guruge, Keerthi S.; Seike, Nobuyasu; Shirai, Yuji; Yamata, Toshiaki; Nakamura, Masafumi; Handa, Hiroshi; Yamanaka, Noriko; Miyazaki, Shigeru. 2007. Determination of PCDD/Fs and dioxinlike PCBs in fish oils for feed ingredients by congenerspecific chemical analysis and CALUX bioassay. Chemosphere. 69:1188–1194.

70

Analysis of Endocrine Disrupting Compounds in Food

Hays, Sean M.; Aylward, Lesa L. 2003. Dioxin risk in perspective: Past, present, and future. Regul. Toxicol. Pharm. 37:202–217. Hayward, Douglas G.; Nortrup, David; Gardner, Albert; Clower, Jr. Marion. 1999. Elevated TCDD in chicken eggs and farm-raised catfish fed a diet with ball clay from a Southern United States mine. Environ. Res. Section A. 81:248–256. Hayward, Douglas G.; Holcomb, Jim; Glidden, Robert; Wilson, Paige; Harris, Mark; Spencer, Virginia. 2001. Quadrupole ion storage tandem mass spectrometry and high-resolution mass spectrometry: Complementary application in the measurement of 2,3,7,8-chlorine substituted dibenzo-p-dioxins and dibenzofurans in US foods. Chemosphere. 43:407– 415. Hess, Philipp; de Boer, Jacob; Cofino, Wim P.; Leonards, Pim E.G; Wells, David E.J. 1995. Critical review of the analysis of non- and mono-ortho-chlorobiphenyls. J. Chromatogr. A. 703:417–465. Hess, Philipp; Wells, David E. 2001. Evaluation of dialysis as a technique for the removal of lipids prior to the GC determination of ortho and non-ortho chlorobiphenyls, using 14C-labelled congeners. Analyst. 126: 829–834. Hoh, Eunha; Mastovska, Katerina; Lehotay, Steven J. 2007. Optimization of separation and detection conditions for comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans. J. Chromatogr. A. 1145:210–221. Hoh, Eunha; Lehotay, Steven J.; Mastovska, Katerina; Huwe, Janice K. 2008. Evaluation of automated direct sample introduction with comprehensive twodimensional gas chromatography/time-of-flight mass spectrometry for the screening analysis of dioxins in fish oil. J. Chromatogr. A. 1201(1):69–77. Hoogenboom, Laurentius A.P.; Goeyens, Leo; Carbonnelle, Sophie; van Loco, Joris; Beernaert, Hedwig; Baeyens, Willy; Traag, Wim A.; Jacobs, Greg; Schoeters, Greet. 2006. The CALUX bioassay: Status of its application to screening food and feed. Trends Anal. Chem. 25:410–420. Hoogenboom, Laurentius A.P.; Eijkeren, Jan C.H.; van Zeilmaker, Marco Jacob; Mengelers, Marcel J.B.; Herbes, R.; Immerzeel, Jaap; Traag, Wim A. 2007. A novel source for dioxins present in recycled fat from gelatine production. Chemosphere. 68:814–823. Jacobs, Miriam; Ferrario, Joseph; Byrne, Christian. 2002. Investigation of polychlorinated-p-dioxins, dibenzofurans, and selected coplanar biphenyls in Scottish farmed Atlantic salmon (Salmo salar). Chemosphere. 47:183–191. Jia, Chang Rong; Qiu, Y. Ming; Ou, Qingyou M. 1991. Multidimensional gas chromatography for the separation of TCDD isomers. Chin. Chem. Lett. 1(2): 31–34. Jiménez, Begoña; Wright, Christopher; Kelly, Mitchum; Startin, James R. 1996. Levels of PCDDs and PCDFs in oil components of the Spanish diet. Chemosphere. 32:461–467. Jones, Jennifer M.; Anderson, Jack W.; Tukey, Robert H. 2000. Using the metabolism of PAHs in a human

cell line to characterize environmental samples. Environ. Toxicol. Pharmacol. 8:119–126. Kiviranta, Hannu; Hallikainen Anja; Ovaskainen, MarjaLeena; Kumpulainen Jorma; Vartiainen, Terttu. 2001. Dietary intakes of polychlorinated dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls in Finland. Food Addit. Contam. 18:945–953. Kiviranta, Hannu; Ovaskainen, Marja-Leena; Vartiainen, Terttu. 2004. Market basket study on dietary intake of PCDD/Fs, PCBs, and PBDEs in Finland. Environ. Int. 30:923–932. Knutzen, Jon; Bjerkeng, Birger; Naes, Kristoffer; Schlabach, Martin. 2003. Polychlorinated dibenzofurans/dibenzo-p-dioxins (PCDF/PCDDs) and other dioxin-like substances in marine organisms from the Greenland fjords, S. Norway, 1975–2001: Present contamination levels, trends, and species specific accumulation of PCDF/PCDD congeners. Chemosphere 52:745–760. Korytár, Peter; Danielsson, Conny; Leonards, Pim E.G.; Haglund, Peter; de Boer, Jacob; Brinkman, Udo A.Th. 2004. Separation of seventeen 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins and dibenzofurans and 12 dioxin-like polychlorinated biphenyls by comprehensive two-dimensional gas chromatography with electron-capture detection. J. Chromatogr. A, 1038: 189–199. Korytár, Peter; Parera, Jordi; Leonards, Pim E.G.; de Boer, Jacob; Brinkman, Udo A.Th. 2005. Quadrupole mass spectrometer operating in the electron-capture negative ion mode as detector for comprehensive twodimensional gas chromatography. J. Chromatogr. A. 1067:255–264. Krokos, Fragoulis; Creaser, Colin S.; Wright, Christopher; Startin, James R. 1997. Congenerspecific method for the determination of ortho and non-ortho polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans in foods by carbon-column fractionation and gas chromatography-isotope dilution mass spectrometry. Fresenius J. Anal. Chem. 357:732–742. Kumar, Kurunthachalam Senthil; Kannan, Kurunthachalam; Paramasivan, Odathurai N.; Sundaram, Vellakovil P. Shanmuga; Nakanishi, Junko; Masunaga, Shigeki. 2001. Polychlorinated dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls in human tissues, meat, fish, and wildlife samples from India. Environ. Sci. Technol. 35:3448–3455. Lamparski, Lester L.; Nestrick, Terry J.; Stehl, Rudolph H. 1979. Determination of part-per-trillion concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxins in fish. Anal. Chem. 51:1453–1458. Lanbropoulou, Dimitra Q.; Konstantinou, Ioannis K.; Albanis, Triantafyllos A. 2006. Sample pretreatment method for the determination of polychlorinated biphenyls in bird livers using ultrasonic extraction followed by headspace solid-phase microextraction and gas chromatography-mass spectrometry. J. Chromatog. A. 1124:97–105. Liu, Hanxia X.; Zhang, Quinghua; Cai, Zongwei; Li, An; Wang, Yawei; Jian, Guibin. 2006. Separation of polybrominated diphenyl ethers, polychlorinated biphe-

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nyls, polychlorinated dibenzo-p-dioxins, and dibenzofurans in environmental samples using silica gel and florisil fractionation chromatography. Anal. Chem. Acta. 557:314–320. Liem, A. K. Djien. 1999a. Basic aspects of methods for the determination of dioxins and PCBs in foodstuffs and human tissues. Trends Anal. Chem. 18:429–439. Liem, A. K. Djien. 1999b. Important developments in methods and techniques for the determination of dioxins and PCBs in foodstuffs and human tissues. Trends Anal. Chem. 18:499–507. Lin, Po-Hsiung; Lin, Chia-Hua; Huang, Chuan-Chen; Chuang, Ming-Chien; Lin, Pinpin. 2007. 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) induces oxidative stress, DNA strand breaks, and poly(ADP-ribose) polymerase-1 activation in human breast carcinoma cell lines. Toxicol. Lett. 172:146–158. Liu, Zaiyou Y.; Phillips, John B. 1991. Comprehensive 2-dimensional gas-chromatography using an oncolumn thermal modulator interface. J. Chromatogr. Sci. 29:227–231. Llobet, Juan M.; Martí-Cid, Roser; Castell, Victoria; Domingo, José L. 2008. Significant decreasing trend in human dietary exposure to PCDD/PCDFs and PCBs in Catalonia, Spain. Toxicol. Lett. 178:117–126. Loran, Susana; Bayarri, Susana; Conchello, Pilar; Herrera, Antonio. 2007. Evaluation of GC-ion trapMS/MS methodology for monitoring PCDD/Fs in infant formulas. Chemosphere 67:513–520. Lund, Kirsten H.; Sørensen, Søren; Cederberg, L. Tommy. 2008. PCDD/F and PCB content in different parts of sheep. Organohalogen Compd. 70:1724– 1727. Lundgren, Kjell; van Babel, Bert; Tysklind, Mats. 2002. Development of a high-performance liquid chromatography carbon column based method for the fractionation of dioxin-like polychlorinated biphenyls. J. Chromatogr. A. 962:79–93. Malavia, Jessica; Ábalos, Manuela; Santos, F. Javier; Abad, Esteban; Rivera, Josep; Galceran, M. Teresa. 2007a. Analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in vegetable oil samples by gas chromatography-ion-trap tandem mass spectrometry. J. Chromatogr. A. 1149:321–332. Malavia, Jessica; Ábalos, Manuela; Santos, F. Javier; Abad, Esteban; Rivera, Josep; Galceran, M. Teresa. 2007b. Ion-trap tandem mass spectrometry for the analysis of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls in food. J. Agric. Food Chem. 55:10531–10539. Malavia, Jessica; Santos, F. Javier; Galceran, M. Teresa. 2008. Comparison of gas chromatography–ion-trap tandem mass spectrometry systems for the determination of polychlorinated dibenzo-p-dioxins, dibenzofurans and dioxin-like polychlorinated biphenyls. J. Chromatogr. A. 1186:302–311. Malisch, Rainer. 2000. Increase of the PCDD/Fcontamination of milk, butter, and meat samples by use of contaminated citrus pulp. Chemosphere. 40: 1041–1053. Matthews, Veronica; Päpke, Olaf; Gaus, Caroline. 2008. PCDD/Fs and PCBs in seafood species from Moreton

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Bay, Queensland, Australia. Mar. Pollut. Bull. 57: 392–402. Mayer, Richard. 2001. PCDD/F levels in food and canteen meals from Southern Germany. Chemosphere. 43:857–860. Miyawaki, Takashi; Makino, Takanori; Honda, Katsuhisa. 2009. Rapid extraction of dioxins in soils and sediments by microwave-assisted extraction (2). Bunseki Kagaku. 58:21–26. Miyawaki, Takashi; Kawashima, Ayato; Honda, Katsuhisa. 2008. Development of supercritical carbon dioxide extraction with a solid phase trap for dioxins in soils and sediments. Chemosphere. 70:648–655. Mondello, Luigi; Tranchida, Peter. 2008. Comprehensive chromatography methods. J. Sep. Sci. 31:3285– 3286. Nording, Malin; Sporring, Sune; Wiberg, Karin; Björklund, Erland; Haglund, Peter. 2005. Monitoring dioxins in food and feedstuffs using accelerated solvent extraction with a novel integrated carbon fractionation cell in combination with CAFLUX bioassay. Anal. Bioanal. Chem. 381:1472–1475. Norwegian Institute of Public Health. Folkehelseinstituttet. Oslo, Norway. Available at: http:// www.fhi.no Ormerod, Steve J.; Tyler, Stephanie J.; Juttner, Ingrid. 2000. Effects of point source PCB contamination on breeding performance and post-fledging survival in the dipper Cinclus cinclus. Environ. Pollut. 110:505–513. Otaka, Hiroaki; Hashimoto, Shunji. 2004. Fast matrix digestion with ethanolic alkali plus pyrogallol for polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls analysis in biological samples. Anal. Chem. Acta. 509:21–26. Papadopoulus, Athanasios; Vassiliadou, Irene; Costopoulou, Danae; Papanicolau, Christina; Leondiadis, Leondios. 2004. Levels of dioxins and dioxin-like PCBs in food samples on the Greek market. Chemosphere. 57:413–419. Pirard, Catherine; Focant, Jean-François; De Pauw, Edwin. 2002. An improved clean-up strategy for simultaneous analysis of polychlorinated diebnzo-pdioxins (PCDD), and polychlorinated dibenzofurans (PDF), and polychlorinated biphenyls (PCB) in fatty food samples. Anal. Bioanal. Chim. 372:373–381. QUASIMEME. Quality Assurance of Information for Marine Environmental Monitoring in Europe. Available at: www.quasimeme.org Ramos, Lourdes; Hernández, Luis Manuel; González, María José. 1997. Elution pattern of planar chlorinated biphenyls and 2,3,7,8-polychlorinated dibenzo-pdioxins/dibenzofurans on chromatographic adsorbents and factors affecting the mechanism of retention possibilities of selective separation of both families. J. Chromatogr. A. 759:127–137. Ramos, Lourdes; Hernández, Luis Manuel; González, María José. 1999. Simultaneous separation of coplanar and chiral polychlorinated biphenyls by off-line pyrenyl-silica liquid chromatography and gas chromatography. Enantiomeric ratios of chiral congeners. Anal. Chem. 71:70–77.

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Ryan, John J.; Lizotte, Raymonde; Panopio, Luz G.; Lau, Benjamin P.Y. 1989. The effect of strong alkali on the determination of polychlorinated dibenzofurans PCDs and polychlorinated dibenzo-p-dioxins PCDDs. Chemosphere. 18:149–154. Ryan, John J.; Conacher, Henry B.S.; Panopio, Luz G.; Lau, Benjamin P.Y; Hardy, Jacques A. 1991. Gas chromatographic separations of all 136 tetra- to octapolychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans on nine different stationary phases. J. Chromatogr. A. 541:131–183. Santillo, David; Fernandes, Alwyn; Stringer, Rowan; Alcock, Ruth E.; Rose, Martin; White, Shaun; Jones, Kevin C.; Johnston, Paul. 2003. Butter as an indicator of regional persistent organic pollutant contamination: Further development of the approach using polychlorinated dioxins and furans (PCDD/Fs), and dioxin-like polychlorinated biphenyls (PCBs). Food Addit. Contam. 20:281–290. Sasamoto, Takeo; Ushio, Fusao; Kikutani, Norihisa; Saitoh, Yuki; Yamaki, Yumiko; Hashimoto, Tsuneo; Horii, Shoxo; Nakagawa, Jun-ichi; Ibe, Akihiro. 2006. Estimation of 1999–2004 dietary daily intake of PCDDs, PCDFs, and dioxin-like PCBs by a total diet study in metropolitan Tokyo, Japan. Chemosphere. 64:634–641. Schecter, Arnold. 1994. Dioxins and Health. Plenum Press, New York, USA. p. 736. Schomburg, Gerhard; Husmann, Heribert; Hübinger, Edward. 1985. Multidimensional separation of isomeric species of chlorinated hydrocarbons such as PCB, PCDD, and PCDF. J. High Res. Chromatogr. 8:395–400. Seidel, Shawn D.; Li, Violet; Winter, Greg M.; Rogers, William J.; Martinez, Eugenio I.; Denison, Michael S. 2005. Ah receptor-based chemical screening bioassays: Application and limitations for the detection of Ah receptors agonists. Toxicol. Sci. 55:107–115. Serrano, Roberto; Fernández, Mario A.; Rabanal, Rosa; Hernández, Luis Manuel; González, María José. 2000. Congener-specific determination of polychlorinated biphenyls in shark and grouper livers from the northwest African Atlantic Ocean. Arch. Environ. Contam. Toxicol. 38:217–224. Smith, Lawrence M.; Stalling, David L.; Johnson, James L. 1984. Determination of part-per-trillion levels of polychlorinated dibenzofurans and dioxins in environmental samples. Anal. Chem. 56:1830–1842. Smith, Roger M. 1999. Supercritical fluids in separation science—the dreams, the reality and the future. J. Chromatogr. A. 856:83–115. Strandberg, Bo; Bergqvist, Per-Anders; Rappe, Christoffer. 1998. Dyalisis with semipermeable membrane as an efficient lipid removal method in the analysis of bioaccumulative chemicals. Anal. Chem. 70: 528–533. Sweetman, Andrew J.; Alcock, Ruth E.; Wittsiepe, Jurgen; Jones, Kevin C. 2000. Human exposure to PCDD/Fs in the UK: The development of a modeling approach to give historical and future perspectives. Environ. Int. 26:37–47. Traag, Wim A.; Immerzeel, Jaap; Onstenk, Constant; Kraats, Cornelius; Lee, Martijn K.; Van der Weg,

Guido; Mol, Hans; Hoogenboom, Laurentius A.P. 2008. Automation of chemical analysis of PCDD/Fs dioxin-like PCBs, indicator PCBs, and polybrominated diphenyl ethers in food and feed. Organohalogen Compd. 70:54–57. Tsutsumi, Tomoaki; Yanagi, Toshihiko; Nakamura, Munetomo; Kono, Yoichi; Uchibe, Hiroyasu; Iida, Takao; Hori, Tsuguhide; Nakagawa, Reiko; Tobiishi, Kazuhiro; Matsuda, Rieko; Sasaki, Kumiko; Toyoda, Masatake. 2001. Update of daily intake of PCDDs, PCDFs, and dioxin-like PCBs from food in Japan. Chemosphere. 45:1129–1137. Tsutsumi, Tomoaki; Miyoshi, Noriko; Sasaki, Kumiko; Maitani, Tamio. 2008 Biosensor immunoassay for the screening of dioxin-like polychlorinated biphenyls in retail fish. Anal. Chim. Acta. 617:177–183. U.S. Environmental Protection Agency (U.S. EPA) 1999. Method 1668. Toxic polychlorinated biphenyls by isotope dilution high resolution gas chromatography/ high resolution mass spectrometry. Washington, DC. U.S. Environmental Protection Agency (U.S. EPA) 2002. Method 1613 Revision B. Tetra-through octachlorinated dioxins and furans by isotope dilution HRGC/HRMS. Washington, DC. van Babel, Bert; Järemo, Mattias; Karlsson, Lars; Gunilla, Lidström. 1996. Development of a solid phase carbon trap for simultaneous determination of PCDDs, PCDFs, PCBs, and pesticides in environmental samples using SFE-LC. Anal. Chem. 68:1279– 1283. van den Berg, Martin; Birnbaum, Linda S.; Denison, Michael; De Vito, Mike; Farland, William; Feeley, Mark; Fiedler, Heidelore; Hakansson, Helen; Hanberg, Annika; Haws, Laurie; Rose, Martin; Safe, Stephen; Schrenk, Dieter; Tohyama, Chiharu; Tritscher, Angelika; Tuomisto, Jouko; Tysklind, Mats; Walker, Nigel; Peterson, Richard E. 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicol. Sci. 93:223–241. van der Velde, Els G.; Hijman, Willie C.; Linders, Sylvia H.M.A.; Liem, A.K. Djien. 1996. SFE as clean-up technique for ppt-levels of PCBs in fatty samples. Organohlogen Compds. 27:247–250. van Ede, Karin; Li, An; Antunes-Fernandes, Elsa; Mulder, Patrick; Peijnenburg, Ad; Hoogenboom, Laurentius A.P. 2008. Bioassay directed identification of natural aryl hydrocarbon-receptor agonists in marmalade. Anal. Chim. Acta. 617:238–245. van Leeuwen, F.X. Rolaf; Feeley, Mark; Schrenk, Dieter; Larsen, John Christian; Farland, William; Younes, Maged. 2000. Dioxins: WHO’s tolerable daily intake (TDI) revisited. Chemosphere. 40: 1095–1101. van Leeuwen, Stefan P.J.; Leonards, Pim E.G.; Traag, Wim A.; Hoogenboom, Laurentius A. P.; de Boer, Jacob. 2007. Polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls in fish from the Netherlands: Concentrations, profiles and comparison with DR CALUX® bioassay results. Anal. Bioanal. Chem. 389:321–333. van Loco, Joris; van Leeuwen, Stefan P.J.; Roos, Peter; Carbonnelle, Sophie; de Boer, Jacob; Goeyens, Leo;

Analysis of Dioxins and Furans (PCDDs and PCDFs) in Food

Beernaert, Hedwig. 2004. The international validation of bio- and chemical-analytical screening methods for dioxins and dioxin-like PCBs: The DIFFERENCE project rounds 1 and 2. Talanta. 63:1169–1182. Vetter, Walter; Weichbrodt, Marion; Hummert, Kerstin; Glotz, Doris; Luckas Bernd. 1998. Combined microwave-assisted extraction and gel permeation chromatography for the determination of chlorinated hydrocarbons in seal blubber and cod livers. Chemosphere. 37:2439–2449. Wang, I-Ching; Wua, Yee-Lin; Linc, Long-Full; ChangChiend, Guo-Ping. 2009. Human dietary exposure to polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in Taiwan. J. Hazard. Mater. 164:621–626. Wiberg, Karen; Sporring, Suni; Haglund, Peter; Björklund, Erland. 2007. Selective pressurised liquid

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extraction of polychlorinated dibenzo-p-dioxins, dibenzofurans, and dioxin-like polychlorinated biphenyls, from food and feed samples. J. Chromatogr. A. 1138:55–64. Wilken, Michael; Martin, Gregory D.; Hescott, Tracy L.; Mendyk, Kendra K.; Fishman, Vyacheslav N; Lamparski, Lester L.; Luksemburg William J.; Maier, Marta; Sünderhauf, W; Van Ryckeghem, Marc; Neugebauer, Frank; de Smet, Geert. 2008. Interlaboratory comparison of the determination of chlorinated dibenzo-p-dioxins and dibenzofurans according to regulatory methods EN 1948 and EPA 1613b. Chemosphere. 73:S2-S6. Wittsiepe, Jürgen; Schrey, Petra; Wilhelm, Michael. 2001. Dairy intake of PCDD/F by small children with different food consumption measured by the duplicated method. Chemosphere 43:881–887.

Chapter 4 Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities M.J. Gómez, M.A. Martínez-Uroz, M.M. Gómez-Ramos, A. Agüera, and A.R. Fernández-Alba Introduction and scope Endocrine-disrupting chemicals (EDCs) can be natural chemicals, but they are mainly man-made. They interfere with the body’s ability to regulate growth, development, metabolism, and other functions. There are hundreds of EDCs in the environment, in food, and in consumer products. They include persistent organic pollutants, pesticides, some heavy metals, preservatives and fragrances, industrial chemicals and their by-products or waste, as well as plant-derived compounds. EDCs can contribute to a wide range of diseases and disabilities, including obesity, diabetes, cancer, heart disease, reproductive health problems, as well as neurodevelopmental and neurodegenerative disorders (Diamanti-Kandarakis et al. 2009). Effects on reproduction and on the immune system have been reported in fish, alligators, seals, and birds (EC-BKH 2000), but evidence for the effects on humans are varied and sometimes contradictory. EDCs have been highlighted as a result of the publication of Our Stolen Future (Colborn et al. 1996) and Assault on the Male (Twombly 1995), which warned of the health effects of many chemicals suspected of interfering with the endocrine systems of both humans and wildlife. This was the case with dichlorodiphenyl trichloroethane (DDT) following the Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

publication of the book Silent Spring in 1962 (Carson 1962), which warned of DDT’s threat to ecosystems. Since the publication of Our Stolen Future, the general public has become worried about the health risks connected to EDCs and proper regulations for EDCs have become requirements for governments. EDCs are sometimes referred to as estrogen mimics. The incidence of cancer is increasing steadily in tissues that are regulated by estrogens. It has been proposed that not only increased cancer incidence, but also fertility problems in humans and wildlife are caused by an increased exposure to compounds that mimic endogenous estrogens (Boenke et al. 2002). Hormones work in the body in very tiny amounts, so even small exposures to EDCs can affect development and function. Unlike most other substances, low doses of EDCs may be more harmful than higher doses. Higher exposures may overwhelm the endocrine system and cause less of a response to the chemical (Diamanti-Kandarakis et al. 2009). This is a concern because safety testing of chemicals assumes that low doses are not harmful if higher doses do not show effects. Most EDCs have not been thoroughly tested for their health effects at very low exposures (Diamanti-Kandarakis et al. 2009). Endocrine-disrupting chemicals can be found in different places and in different products throughout the world. For most of the reviewed compounds, food is the main EDC exposure route for humans. Because the 75

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chemicals are so widespread, and considering the vast amount of different potential endocrine disrupters used in food, even if human exposure to a single endocrine-disrupting chemical is considered to be low, chronic exposure to EDCs is toxicologically relevant. Concerns increase when humans are exposed to mixtures of similar-acting EDCs and/or during sensitive windows of development (fetuses and children). Hence, potential additive or synergistic effects have to be considered when assessing human risk (Yang et al. 2006). Fish and seafood from contaminated water can have EDCs in their flesh and fat. EDCs bioaccumulate in animals: they are found in higher concentrations in animals that eat other animals. Food can also be contaminated through the use of pesticides that have endocrine-disrupting properties. Pesticides are widely used to combat diseases and pests and may adversely affect the production of vegetable and animal foodstuff; they comprise a large number of substances that belong to different chemical groups. Pesticides can be classified based on functional groups in their molecular structure (e.g., inorganic, organonitrogen, organohalogen, or organosulfur compounds) or their specific biological activity on target species (e.g., insecticides, fungicides, herbicides, acaricides, etc.) The use of these compounds in agriculture has progressively increased after World War II, leading to increased world food production. Nevertheless, this use and the additional environmental pollution due to industrial emission during their production have resulted in the occurrence of residues of these chemicals and their metabolites in food commodities, water, and soil (Ahmed, 2001). Their adverse effects on human health may include acute neurological toxicity; chronic neurodevelopment impairment; possible dysfunction of the immune, reproductive, and endocrine systems; cancer, and many other adverse effects (Hercegová et al. 2007).

To protect the environment and consumers’ health, many countries have restricted the use of pesticides and have established legal directives to control their levels in food by establishing maximum residue levels (MRLs). The determination of pesticide residues is a requirement to support the enforcement of legislation, ensure trading compliance, and conduct residue monitoring programs in dietary components and in environmental samples (Fong et al. 1999). Organochlorine pesticides (OCPs), effective against a variety of insects, have been extensively used around the world. Their low volatility, combined with their extreme stability and probable indiscriminate use in the past, has led to their persistence both in the environment after application and in organisms after exposure. A major characteristic of some of these compounds is their ability to accumulate and concentrate in biological systems. Because of slow breakdown and elimination, their residues can remain for long periods in human and animal tissue. Organochlorine endocrine-disrupter pesticides (ED pesticides) have been of great concern due to their persistent nature and chronic adverse effect on wildlife and humans. Despite the ban and restriction on the use of organochlorine ED pesticides in developed countries during the 1970s and 1980s, some developing countries are still using them for agricultural and public purposes because of the low cost and versatility in controlling various insects. OCPs are well-known as endocrine disrupters; in fact, the ability of pesticides to interact with the endocrine function has been known for decades due to many organochlorine insecticides that can interact with estrogen, androgen, and progesterone receptors. DDT, or 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane, is the first of the chlorinated organic pesticides. It has been one of the most used and more persistent of this group of compounds. Commercial DDT comprises several isomers, the most prevalent being the p,p′-form, which makes up about 75%–

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

80% of the total. The ability of the prevalent isomer of the major and most persistent DDT derivative, p,p′dichlorodiphenyldichloroethylene (p,p′DDE), to bind to the androgen receptor in male rats has been reported (IEH 1995). Organochlorine pesticides have also been linked to human breast and liver cancers and to testicular tumors and lower sperm counts in humans (IEH 1995). DDT and its analog, DDE, are the archetypes of fat-soluble, nonbiodegradable, and bioaccumulating compounds. Even in countries across North America and Europe, where its use has been banned for over a decade, DDT and DDE residues are still often found in food. This is because of environmental persistence, illegal use, or importation of contaminated food from regions where DDT is still used. Other organochlorine pesticides classified as endocrine disrupters are hexachlorocyclohexane (HCH), chlordane, aldrin, dieldrin, endrin, endosulfan, heptachlor, hexachlorbenzene (HCB), and dicofol. In addition to OCPs, there are some chlorine herbicides such as acetochlor, alachlor, atrazine, simazine, linuron, diuron, nitrofen, and 2,4-D; chlorine fungicides such as vinclozolin, iprodione, prochloraz, triadimefon; and the pyrethroid cypermethrin (Table 4.1) that are also classified as known or suspected endocrine disruptors. Unlike traditional OCPs, most of these ED pesticides are widely used today and are more polar compounds. We will refer to all these compounds as organochlorine endocrinedisrupter pesticides. The global increase in concern regarding ED pesticides has made it necessary to develop highly sensitive and specific analytical tools to measure their presence in food samples far below the established maximum residue limits (MRLs). Revision and optimization of existing methods are also needed in order to have reliable tools for risk assessment and control of human exposure to these compounds.

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This chapter aims to offer an overview of the state of the art of the analytical methodologies developed over the last few years for the analysis of organochlorine ED pesticides in food samples, including sample preparation and detection. Several reviews have been published about the determination of pesticides and other groups of toxic substances in food samples, but not many focus on EDCs. In addition to the review of the procedures described in the literature for the analysis of the chlorinated ED pesticides, the occurrence of these compounds reported in the studied matrices will be discussed.

Endocrine-disrupter categorization In recent years, various international and national governmental bodies and nongovernmental organizations have published lists of suspected EDs. The Institute for the Environment and Health (IEH 2005) has developed a database of these lists, together with further data extracted from original published studies or review articles. This database contains a total of 966 compounds or elements purported to be EDCs. One of the sources used in the compilation of the IEH database is the BKH Consultants’ report from the European Commission, entitled Towards the Establishment of a Priority List of Substances for Further Evaluation of Their Role in Endocrine Disruption (ECBKH 2000). It is a list of 553 anthropogenic chemicals known or suspected to be EDs, prioritized in terms of their potential for human and wildlife exposure, their high production volumes (HPV) and/or persistence, and the strength of scientific evidence regarding their having endocrine properties. In this way, they are classified into three groups: Group I. Compounds for which there is considered to be evidence of endocrinedisrupting activity and for which a high level of concern exists with regard to exposure.

78 Insecticide mostly on nonfood crops (used to control termites, and on home lawns and gardens); forbidden in EU and US Stable metaboliteoxychlordane. Insecticide banned in all countries for use in agriculture. Still used in disease vector control (e.g., malaria); recommended for indoor spraying only. Insecticide on seed and soil before food crops are planted. Also used as a timber treatment and in the home for head lice.

Organochlorines

Fungicide on seeds and food crops; severely restricted in the EU but still used in some parts of the world.

Use

Organochlorine EndocrineDisrupter Pesticides Vegetable oils, fish oil, pork fat, eggs, meat, milk, cheese, butter, tea, cocoa, coffee, vegetables, fruits; found in mother ’s milk; due to atmospheric transport, Inuit women tend to have a diet highly contaminated with chlordane. Fruits, vegetables, rice, eggs, milk, butter, fish fat; widespread persistence in environment, biota, mother ’s milk and food. Exposure has occurred due to eating contaminated fish and animals. Olive oil, vegetables, fruits, milk, butter, cheese, meat, chocolate, cereals, baby food; long-range transport seen. Observed in biota and water systems, through wastewater at production, and through application on soil and seeds. Vegetable oils, fish oil and fat, pork fat, vegetables, fruits, rice, eggs, baby food, milk, butter; long-range transport. Found in environment and biota. Exposure through production at industrial wastewater.

Target Matrices/Human Exposure

Hexachlorobenzene (HCB)

Hexachlorocyclohexane isomers (HCHs) Lindane (gamma-HCH)

DDT and metabolites

Chlordane

Chemical Contaminant

Table 4.1. Information for organochlorine endocrine-disrupter pesticides.

Cl

Cl Cl

Cl

Cl

Cl

Cl Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Cl

Structures

Cl

Cl

Group I Persistent, bioaccumulated High production volumea

Group I Inherently biodegradable, bioaccumulated High production volumea

Group I Persistent, bioaccumulated High production volumea

Group I Persistent, bioaccumulation observed

Notes

79

Dicofol

Heptachlor

Vegetables, fruits; exposure through contaminated fruits and vegetables. Found in human body fat.

Vegetable oils, fish oil and fat, pork fat, fruits, vegetables, eggs, milk, butter, baby food, meat; exposure through contaminated vegetables, fruits and animals (through wastewater at production and application)

Acaricide Can be contaminated with alpha-Cl-DDT. In EU, dicofol is not permitted if it contains less than 78% of pp dicofol or more than 1 g/kg DDT and related compounds. Insecticides

Endosulfan (alpha, beta, and sulfate)

Olive oil, olives, vegetables, fruits, fruit juices, eggs, baby food, butter, milk; exposure through contaminated animals, fruits and vegetables.

Insecticide and acaricide; widely used in developing countries

Chemical Contaminant

Organochlorines

Target Matrices/Human Exposure

Use

Organochlorine EndocrineDisrupter Pesticides

Cl

Cl

Cl

O

O

Cl

Cl

Cl

Cl

Cl

Cl

Cl Cl

Cl

O

S

O

Cl

Cl

O

Cl

Cl

Cl

Cl

O S O

O

O S O

Cl

OH

Cl

Cl H

Cl Cl

Cl H Cl H

Cl Cl

Cl H

Structures

Cl

Cl

Group II

Group II

(continued)

Group II Persistent, bioaccumulated

Notes

80

Organochlorine EndocrineDisrupter Pesticides Herbicides

Aldrin

Endrin

Aldrin can break down to dieldrin.

Endrin, also avicide and rodenticide.

Chemical Contaminant

Dieldrin

Target Matrices/Human Exposure

Dieldrin banned

Use

Table 4.1. Information for organochlorine endocrine-disrupter pesticides. (cont.)

O

O

H

H

Cl

Cl

Cl

Cl

Cl

Cl

Cl Cl

Cl

Cl

H Cl

Cl H

Cl

Cl

Cl

Cl

Cl

Structures

Cl

Group II

Group II

Group II Persistent, bioaccumulated

Notes

81

Herbicides

Organochlorine EndocrineDisrupter Pesticides Vegetables, fruits, rice, milk, meat; exposure through contaminated vegetables, fruits, and animals.

Olive oil, butter, fruits, fruit juices, vegetables, eggs, raisins, milk, baby food, meat; contaminated soil alongside roads. Exposure through contaminated vegetables, fruits and animals.

Triazine herbicide on food crops and alongside roads and uncultured land.

Target Matrices/Human Exposure

Amide herbicide also on food crops.

Use

Simazine

Atrazine

Alachlor

Acetochlor

Chemical Contaminant

H3C

H3C

N

Cl

N H

N H

N

CH3 N

Et O

N

CH3 Et

O CH3

Cl

N

Cl

N

CH3

N

N

Cl

N H

N H

CH3 O

O

Structures

CH3

CH3 Group II

(continued)

Group I Persistent High production volumea

Group I Slightly bioaccumulated High production volumea

Group I Slightly bioaccumulated High production volumea

Notes

82

Organochlorine EndocrineDisrupter Pesticides Herbicides

Target Matrices/Human Exposure

Olive oil, fruits, vegetables, raisins, cereals, meat; exposure through contaminated vegetables, fruits and animals (through wastewater at production and application)

Vegetables, eggs; exposure through contaminated vegetables and animals.

Human milk, barley; exposure through contaminated vegetables and animals.

Use

Urea herbicides on food crops Linuron biodegradable into metabolite 3,4-DCA

Diphenyl ether herbicide

Phenoxy herbicide

2,4-Dichlorophenol (2,4-D) Pesticide intermediate

Nitrofen

Diuron

Linuron

Chemical Contaminant

Table 4.1. Information for organochlorine endocrine-disrupter pesticides. (cont.)

O

N+

O–

Cl

Cl

OH

Cl

Cl

Cl

Cl

N

O

Cl

NH

O

H3C

H N

N

O

O

Structures

CH3

CH3

CH3

Cl

Group II

Group II

Group II

Group I High production volumea

Notes

83

Olive oil, olives, butter; exposure through contaminated food.

Fruits, vegetables, wheat flour, wine, rice; exposure through contaminated vegetables and fruits.

Fungicide on fruits and food crops

Insecticides widely used in agriculture.

Fruits, vegetables; exposure through contaminated vegetables, fruits, and animals (through wastewater at production and application)

Target Matrices/Human Exposure

Fungicide on fruit, vegetables, and ornamental plants

Use

Cypermethrin

Triadimefon

Prochloraz

Iprodione

Vinclozolin

Chemical Contaminant

O

Cl

Cl

Cl

Cl

N

N

N

O

O

O

N

N

O

CH3

CH3

N

CH3 CH3 CH3

N

H N

Cl

O

CH3

N

O

O

Cl

Cl

O H3C

N

O

O

N

H3C Cl

H2C Cl

H3C

O

Cl

Structures

O

Group III

Group II

Group II

Group II

Group I High production volumea

Notes

Sources: Institute for Environment and Health (IEH); BKH report for the European Commission (EC-BKH). Chemicals in bold type are considered to be substances having evidence of endocrine disruption and are, therefore, of high exposure concern. PCDF, pentachlorodibenzofuran; PCDD, pentachlorodibenzodioxin; TCDD, tetrachlorodibenzo-p-dioxin. a The selection of high production volume chemicals was based on the HPV list from Regulation (EEC) No. 793/93 on chemicals with a production volume of more than 1,000 tons per year.

Pyrethroids

Fungicides

Organochlorine EndocrineDisrupter Pesticides

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Analysis of Endocrine Disrupting Compounds in Food

Group II. Potential endocrine disrupters or compounds for which there was a medium level of concern with regard to exposure. Group III. Compounds for which there is considered to be insufficient evidence of endocrine disruption or for which there is only a low level of concern with regard to exposure.

General strategies for the analysis of endocrine-disrupter pesticides in food commodities

The categorization into high, medium, and low concern was based on qualitative criteria. Chemicals with a high exposure concern are those in which human exposure is expected due to environmental concentrations and presence in food or consumer products and/ or in which wildlife exposure is expected due to use and emission patterns, where the chemical is persistent and bioaccumulative. Chemicals with medium exposure concern are those for which human exposure is not expected but for which wildlife exposure is expected due to use and emission patterns, where the chemical is readily biodegradable and not bioaccumulative. Chemicals with low exposure concern are those for which no human or wildlife exposure is expected. An overview of the organochlorine ED pesticides studied in this chapter is provided in Table 4.1. Information is given regarding use, food matrices in which the compounds have been found, and human exposure routes. Evidence on endocrine disruption, bioaccumulation, and persistence of the individual chemicals is also summarized. The EDCs with high production volume (Regulation [EEC] No. 793/93) are indicated in the table. This is an indicator of exposure probability, because if a chemical is produced in a high enough quantity, there is more likelihood that there is human exposure (ECBKH 2000). The chemicals classified in Group I (in bold) are considered to be substances having evidence of endocrine disruption and are, therefore, of high exposure concern (i.e., humans and wildlife are exposed to them, and they are persistent and bioaccumulative chemicals).

Sample preparation

Food samples are very complex matrices, and the determination of microcontaminants at very low concentrations within them is a difficult task.

Sample preparation is one of the most important steps within an analytical methodology. Although there are thousands of methods for extracting and isolating toxic compounds from food, extraction still represents an analytical challenge. To achieve the low limits of detection required for the detection of EDCs in food samples, a trace-enrichment step is necessary. ED pesticides have been analyzed in a wide variety of food matrices, including solid and liquid samples. Determination of EDCs in these matrices often requires extensive sample preparation prior to instrumental analysis, constituting a bottleneck in food analysis. Sample preparation is generally matrix and analyte dependent, and it is difficult to establish general trends. The extraction methods are selected considering solid and liquid matrices. In general, solid samples, which are the more common in food analysis, require more complex and time-consuming treatments than do liquids: it is necessary to replace the solid matrix with a liquid one. The typical food sample preparation steps include pretreatment, extraction, cleanup, and concentration (Figure 4.1). Sample pretreatment It is often necessary to pretreat solid samples by sieving, grinding, and drying. Dispersion can be used to avoid the aggregation of sample particles and ensure good solvent penetration. Drying is particularly important when using nonpolar solvents because any

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

SOLID FOOD

85

LIQUID FOOD

Fruits, vegetables, cereals, baby f ood, meat, f ish...

Juice, milk, oil, beverages…

PRETREATMENT Homogenization, sieving, grinding, freeze-drying, drying with Na2SO4, dispersion

Filtration, centrifugation, NaCl addition, degassed

EXTRACTION SE, MAE, PLE, Sonication, Soxhlet, SFE, MSPD

LLE, SPE, SPME, SBSE

CLEAN-UP SPE, DSPE,LLE, GPC,column chromatography, Low temperature fat precipitation INSTRUMENTAL ANALYSIS

GC MS,MS/MS, HRMS

LC ECD

MS,MS/Ms HRMS

UV,DAD

Figure 4.1. Scheme of analytical methodologies employed for the determination of organochlorine ED pesticides in food commodities.

moisture can reduce the extraction efficiency, and desiccants such as sodium sulfate, diatomaceous earth, or cellulose can help overcome this. Liquid samples are filtered and/or centrifuged and then handled by solvent–solvent extraction methods or sorption methods; however, special treatments may be required, depending on the matrix composition. Carbonated beverages are degassed. Canned foods containing both liquid and solid portions are usually filtered and treated separately (Ballesteros-Gómez et al. 2009). Some samples, such as animal or fish tissue, can cause special problems. Drying before extraction may not always be practical because of the bulk of the sample. Tissue matrices, in particular, cause problems because they tend

to clump, preventing penetration by the extraction media. Samples with high protein content may require their removal by precipitation. Determination of ED pesticides in food is often complicated by the presence of varying fat content. There are some differences in the analysis of low-fat food such as milk, with a fat content <3%, and high-fat matrices, such as animal fat, butter, or vegetable oils (>20% lipids). Probably the most complex matrices are those with high fat content because it is quite complex to extract the pesticides without coextraction of lipids, which are usually difficult to remove (GilbertLopez 2009). If the lipids are not removed in the sample preparation processes, these nonvolatile compounds can be injected into the chromatographic system, resulting in lower

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Analysis of Endocrine Disrupting Compounds in Food

sensitivity, shorter column life, and possible harm to the detection system. Sample preparation methods are developed taking into consideration the fat content. Extraction of endocrine-disrupter pesticides from solid food matrices Solvent extraction To date, solvent extraction (SE), alternatively called liquid–solid extraction (LSE), is the main technique used for the isolation of ED pesticides, including organochlorine ED pesticides, from solid foodstuffs, mainly because of its simplicity and wide-ranging applicability. The large volume of solvents and the lack of automation are among the main pitfalls of this methodology. Because of the limited selectivity of solvent-based extractions, there is a need for extensive cleanup prior to instrumental analysis. In the last few years, the trend in extraction methods has leaned toward the development of multiresidue methods to control a large number of compounds. Today, there are various well-known and efficient general extraction procedures based on acetonitrile, ethyl acetate, or acetone that can be applied for the simultaneous extraction of a high number of compounds from food matrices (Gilbert-Lopez 2009). Worldwide, two extraction procedures have been applied in pesticide residue analysis in fruit and vegetables: acetone followed by partitioning with a mixture of dichloromethane and light petroleum (the Luke method) and extraction with ethyl acetate in the presence of sodium sulfate. Both established methods have been modified in many aspects. From the point of view of health and environmental safety, the negative impact of ethyl acetate and acetone is much lower than in the case of chlorinated solvents (such as dichloromethane). This was the reason for the modification of the Luke extraction method and consequently, dichloromethane partition was gradually replaced by extraction with, for example, ethyl acetate– cyclohexane (1:1, v/v).

Recently, a quick, easy, cheap, rugged, and safe (QuEChERS) method has become very popular as a powerful procedure in pesticide residue analysis in foodstuffs. Although a very new method (first reported in 2003; Anastassiades et al. 2003), it has already been widely accepted by the international community of pesticide residue analysts, and many publications already deal with this method in its original form or variations of it. This methodology allows the simultaneous extraction of polar and nonpolar compounds with adequate recoveries, and thus it is suitable for the extraction of a wide range of compounds. This method is based on liquid–liquid partitioning with acetonitrile, followed by a dispersive solid-phase extraction (SPE) cleanup step with primary secondary amine (PSA). Acetonitrile is easily and effectively separated from water upon addition of salt. In the QuEChERS method, the addition of anhydrous NaCl and MgSO4 influences liquid–liquid partitioning, providing phase separation without dilution and high recoveries, including rather polar compounds. The main advantage of this protocol is the relatively low solvent consumption, at least as compared to the traditional methods. Soxhlet extraction Soxhlet extraction is another technique that has been widely used in the analysis of solid food samples. The extraction temperature is higher than room temperature, and the sample is repeatedly brought into contact with fresh solvent. The disadvantages of this technique are that it requires large amounts of solvent, the solvent must be evaporated to concentrate analytes before determination, and the process consists of a single sample run that takes many hours to complete. An improved version of the Soxhlet apparatus, which allows for shortening of the extraction time to as little as 1 hour, with a simultaneous reduction of solvent usage, is Soxtec. Extraction in this apparatus is a two-step procedure involving a

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

boiling and a rinsing step, which drastically reduces the total time of extraction. The Soxhlet technique, although exhaustive, is not selective, and further cleanup is necessary (Ridgway et al. 2007). Sonication Sonication is another conventional technique that requires the application of a solvent. This technique applies acoustic energy to enhance food washing. Most of these techniques demand long extraction times and large volumes of hazardous organic solvents, generating dirty extract that requires extensive clean-up before analysis. The extraction methods of recent years are moving toward the use of smaller initial sample sizes, a more environmentally friendly and safer approach (reduction in the total volume of organic solvents consumed), and a faster and less laborious approach to simultaneous preservation of high recoveries and good precision. The main key to shorter extraction times and reduced solvent consumption is the possibility of working at elevated temperatures, above the boiling point of the solvent. These, techniques include microwave-assisted extraction (MAE), pressurized liquid extraction (PLE), also called accelerated solvent extraction (ASE), as well as supercritical fluid extraction (SFE). The extraction process is facilitated in these techniques by the increased analyte desorption and diffusion from the solid matrix. Microwave-assisted extraction MAE is based on the application of microwave energy to the sample during extraction, which is consequently agitated and heated quickly. The main advantages of microwave pretreatment are the low temperature requirement, high extraction rate, automation, and the possibility of simultaneously extracting different samples at the same time without interference. This technique has proven to be better than Soxhlet extraction by cutting

87

solvent consumption and extraction time (Diagne et al. 2002). Accelerated solvent extraction One of the latest contributions to the increasing number of extraction techniques is ASE. This technique uses the decreased viscosity of solvents at higher temperatures in order to enhance the solubility of the target analytes. Pressure keeps the solvent below its boiling point and forces its penetration into the pores of the sample. The combined use of high pressure and temperature provides a faster extraction process that requires smaller amounts of solvent as compared with traditional techniques such as Soxhlet extraction or liquid– liquid extraction (LLE). The disadvantage is the use of expensive and specialized equipment. The major problem with fatty matrices is the presence of large amounts of coextracted lipids, which means that post-cleanup of the extract is required to carry out lipid elimination. To overcome this drawback, simultaneous extraction-purification methods have been developed using a retainer sorbent, such as alumina, Florisil, or sulfuric acid impregnated in silica gel inside the ASE cell (Björklund et al. 2001). Supercritical fluid extraction Another alternative to traditionally used techniques is SFE. The solvent used in this technique is a supercritical fluid (a substance above its critical temperature and pressure that diffuses through solids like a gas, but dissolves analytes like a liquid), usually carbon dioxide. The solvation power of the fluid can be manipulated by changing pressure or temperature and by adding modifiers, such as methanol. A major advantage of using carbon dioxide is that it is environmentally friendly and easily removed from the sample matrix after extraction. The presence of water and fat in food samples can require extensive sample preparation and cleanup. SFE is not used frequently in

88

Analysis of Endocrine Disrupting Compounds in Food

food analyses, probably because of the high cost of instrumentation and the demand for optimization of high numbers of parameters for each matrix. The greatest advantage of SFE in food analysis is the possibility of high extraction selectivity: the obtained extract is relatively pure and, furthermore, is preconcentrated. Matrix solid-phase dispersion Another way of extracting analytes is by using a solid adsorbent material. A sorbent with strong affinity toward some target analytes will retain and concentrate those compounds from the sample solution. Matrix solid-phase dispersion (MSPD) has also been used in the determination of ED pesticides from solid, semisolid, and highly viscous food products. This technique comprises sample homogenization, cellular disruption, fractionation, and purification in a single process. In MSPD, a fine dispersion of the matrix is mixed with a sorbent material (C18, C8, Florisil, silica, etc.) using a mortar and pestle. The more polar phases, such as Florisil, are employed to isolate more polar analytes, whereas the applications using reversed-phase materials have been aimed to isolate more lipophilic compounds. After blending, the sorbent material is often packed into a SPE minicolumn, where the analytes are eluted by a relatively small volume of a suitable eluting solvent. The extracts obtained are generally ready for analysis, but if necessary, they can be easily subjected to direct extract purification by using “co-column” material (e.g., Florisil, silica) that is packed into the bottom of the same column of the sorbent, cleaning the sample as it elutes from the MSPD sorbent–matrix mixture. MSPD efficiency depends on careful optimization of the experimental parameters affecting competition within the matrix, the dispersant sorbent, and the extraction solvent for both analytes and potential matrix interferences. The main advantages of the MSPD procedure

compared to other extraction techniques are that this method is simple and short, the possibility of emulsion formation is eliminated, the solvent consumption is substantially reduced, and the extraction efficiency of the analytes is enhanced as the entire sample is exposed to the extractant. The main disadvantage is the lack of procedure automation. This extraction method has been used to extract pesticides, including organochlorine ED pesticides, in fruits and vegetables and also fatty matrices such as olives, meat, milk, eggs, and fat (Ferrer et al. 2005; Hercegová et al. 2007).

Extraction of endocrine-disrupter pesticides from liquid food matrices Liquid–liquid extraction (LLE) For aqueous samples, LLE is traditionally one of the most common methods of extraction. It is based on the relative solubility of an analyte in two immiscible phases and is governed by the equilibrium distribution/ partition coefficient. Typically, a separating funnel is used, and the two immiscible phases are mixed by shaking and then are allowed to separate. To avoid emulsions, in some cases salt may be added, and centrifugation can be used if necessary. There are many inherent disadvantages with this technique: it is laborious and time consuming; it is apt to form emulsion; and it needs large volumes of organic solvents. Also, due to the limited selectivity, particularly for trace level analysis, cleanup or analyte enrichment and concentration steps are needed prior to instrumental analysis. Removal of lipids from the extract is essential for samples of animal origin (e.g., fish, meat) or fatty vegetable matrices (e.g., olive oil) because they can significantly reduce the analytical performance of liquid chromatography (LC) and gas chromatography (GC). Fat removal is mainly made by LLE combined with lowtemperature fat precipitation.

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

Solid-phase extraction SPE has developed as an alternative to LLE for the separation, purification, concentration, and solvent exchange of solutes for solutions. Today, SPE is a widely used technique for both the extraction of organochlorine ED pesticides contained in liquid foods and the cleanup of crude extracts after solvent extraction or LLE. SPE involves the use of disposable cartridges and disks to trap analytes. As the sample solution passes through the activated sorbent bed, analytes concentrate on its surface, and the other sample components pass through the bed (or vice versa, if necessary for cleanup). Among the advantages of SPE over LLE are higher precision and throughput, lower solvent consumption, and avoidance of the formation of emulsions. In addition, SPE prepares multiple samples in parallel. Added to this, SPE can be easily incorporated into automated analytical procedures, which can lead to greater accuracy and precision and also higher laboratory throughput. On the other hand, concentration factors, enhanced by solvent evaporation, are similar for SPE and LLE. The SPE of liquid foods usually requires further cleanup, and great care must be taken regarding the small particulates present in the sample extracts, which can produce low recoveries and irreproducibility by adsorption of analytes or by clogging. An important aspect to consider in SPE is the selection of the sorbent. A wide range of sorbents have been used for the extraction of ED pesticides from food samples, including C8- and C18-bonded phases on silica; polymeric resins (polystyrene/divinylbenzene copolymer); Florisil (activated magnesium silicate); polar sorbents such as alumina, charcoal, and silica; and cyano- and aminobonded sorbents. Ionic functional groups, such as carboxylic acid or amino groups, can also be bonded to silica or polymeric sorbent to create ion-exchange sorbents. These different phases enable interactions based on adsorption, H-bonding, polar and nonpolar

89

interactions, cation and anion exchange, or size exclusion, where used. The use of the optimum SPE cartridge can have significant effects on recoveries. Selectivity of stationary phases is a key parameter, taking into account the development of selective and sensitive methods for trace analysis. A variety of highly selective SPE sorbent materials have been developed that are especially suitable for the determination of trace contaminants in complex samples such as foods, thus performing extraction and cleanup in one step, as is the case for immunosorbents, restricted access media (RAM), and molecularly imprinted polymers (MIPs). Their application in food analysis is emerging but rather limited so far, perhaps because the development of these materials is time-consuming, complex, and expensive. On the other hand, for multiresidue methodologies that determine simultaneously different groups of contaminants of widely varying structures, the universality of the sample preparation step is necessary, and nonselective hydrophobic sorbents are used for this purpose. The divinylbenzene/ N-vinylpyrolidone copolymer (Oasis HLB) and chemically bonded reversed-phase silica (C18) are the most commonly used sorbents for the isolation of ED pesticides from food commodities. Oasis HLB offers advantages over the classical silica sorbents; for example, high specific area, possibility of drying out during the extraction procedure without reducing its ability to retain the compounds, and like other polymeric resins, stability over the entire pH range. A drawback of this technique is that the packing of the adsorbent must be uniform to avoid poor efficiency, and although prepacked commercial cartridges are now considered reliable, automated systems can have difficulties with reproducibility for some sample types. Over the past decades, the development of automated and miniaturized sample preparation methods with reduced or eliminated

90

Analysis of Endocrine Disrupting Compounds in Food

solvent consumption has become a dominant trend in analytical chemistry. Miniaturized sorptive extraction techniques such as solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE) have the capability of improving the isolation and cleanup of contaminants from food. These techniques are also an improvement in terms of solvent consumption, automation, and sample-handling reduction. However, their application for the extraction of organochlorine ED pesticides from food is still limited to date. Solid-phase microextraction SPME is an alternative to other extraction methods because it integrates sampling, extraction, concentration, and sample introduction into a single step without using a solvent. Due to complications in quantitation, strong dependence on matrix, and certain practical matters, some quality in the results is sacrificed for speed and ease. The SPME device consists of a fused-silica fiber coated with an appropriate stationary phase, where analytes are adsorbed. The extraction of the target analytes from the sample matrix to the fiber happens either directly with the coated fiber immersed in the liquid sample (direct SPME) or in the vapor phase above the liquid sample (this technique is known as headspaceSPME, or HS-SPME). Factors influencing the extraction steps include fiber type, extraction time, ionic strength, sample pH, extraction temperature, and sample agitation. Variables affecting the desorption steps include temperature, desorption time, and focusing oven temperature. Among the advantages of HS-SPME is the high degree of automation as compared to other, more laborious techniques. The procedure can be completely automated using an autosampler with HS-SPME equipment. Other advantages are the inherent high sensitivity and the absence of solvents and sample pretreatment required, thus minimizing sample manipula-

tion and contamination. The main disadvantages include poor fiber-to-fiberreproducibility as well as poor precision and ruggedness of the determinations. The technique is limited to quite volatile compounds. Stir-bar sorptive extraction SBSE is a recent technique based on the sorptive interaction of the compounds of interest with a coat of polydimethylsiloxane (PDMS) deposited on magnetic glass. The extraction mechanism and advantages are similar to those of SPME, but the enrichment factor, which is determined by the amount of extractive phase, is up to 100 times higher. Therefore, SPME is considered ideally suited for the detection of compounds that present high concentrations, whereas SBSE is the method of choice for trace analysis. Transfer of the analyte from the bar is achieved either by GC thermal desorption or elution with an LC solvent. As with SPME, the stir bar can also be used to sample the volatiles and semivolatiles in the headspace above the sample (headspace sorptive extraction, or HSSE). SBSE can be used for liquid or semisolid complex matrices and therefore has potential for many applications in food analysis. SBSE, like SPME, is suitable for the isolation of less-polar compounds; however, this technique can be used for more-polar compounds using derivatization. Applications of SBSE in food analysis are increasing, but due to the limitations of the PDMS phase applications are still currently limited to non-fatty food matrices and nonpolar or semipolar analytes. Other phases are under development and have been tested, as well (David and Sandra 2007). Cleanup methods During the extraction of food samples, not just analytes are isolated. There are different types of interfering compounds, mainly fats, carbohydrates, water, chlorophyll, and others, that are coextracted.

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

After the extraction step, the obtained extracts can be injected directly into the chromatographic system. This is possible using advanced techniques such as comprehensive two-dimensional gas chromatography (GCxGC). This powerful technique can separate analytes of interest from each other and/ or the matrix background better than can conventional one-dimensional gas chromatography (1D-GC), which generally does not provide sufficient separation for highly complex mixtures, as is the case with foodstuffs (Dallüge et al. 2002). However, for complex matrices such as food, the sample preparation methods use different cleanup steps, and the objective of any analytical method should be to achieve the required performance (e.g. sensitivity, accuracy, and precision) in as few steps as possible. The most commonly reported cleanup methods in the analysis of organochlorine ED pesticides in food are LLE, SPE, and GPC. Many cleanup procedures employ SPE using Florisil (magnesium silicate), silica, neutral alumina, or a combination. For some applications these sorbents have also been modified (e.g., sulfuric acid). Florisil is the most commonly applied sorbent and is particularly suited for fatty foods. All of these materials function as polar sorbents, which strongly retain the polar lipids when eluted with organic solvents of relatively low polarity. These adsorption methods are, therefore, most useful for analytes of relatively low polarity, such as many organochlorine pesticides, because more polar analytes will coelute with the lipid fraction. For cleanup of more polar ED pesticides, such as some chlorine fungicides and herbicides, nonspecific hydrophobic sorbents such as charcoal or graphitized carbon black (GCB) are used. Another type of SPE column able to remove nonpolar coextractives that is widely applied for the cleanup of contaminants in different food commodities is reversed-phase C18 sorbent. Oasis HLB is also used to remove hydrophilic and lipophilic interference from

91

food. All these sorbents, although useful for particular classes of ED pesticides, were found to be inadequate for cleanup of matrices of a diverse list of compounds (Gilbert et al. 2009). Chemical-bonded stationary phases such as aminopropyl (–NH2), primarysecondary amine (PSA), and strong anion exchanger (SAX) have been widely applied in multiresidue food analysis. Recent sample treatment protocols focus on the use of a combination of two or three of the commercially available SPE sorbents (GCB, C18, SAX, PSA, −NH2). Gel permeation chromatography (GPC) is generally recommended for the fractionation and/or cleanup of complicated matrices. It can be used for separating large molecules (e.g., lipids), because its principle of operation is based on size exclusion. The divinylbenzene-linked polystyrene gel is the most widely used sorbent for GPC. The procedure can be automated and has mainly been applied to purify fat-rich extracts of both plant and animal origin (García-Reyes et al. 2007; Beyer and Biziuk 2008). GPC has proven to be a universal cleanup procedure in multiresidue methods (MRMs) and is one of the more widely used techniques in routine laboratories for the analysis of pesticide residues in vegetable oil. The acquisition and maintenance costs of the instrument and the large amount of toxic solvents consumed per analysis are important drawbacks of this methodology. Some compounds with high molecular mass (e.g., pyrethroids) need to be sufficiently separated from a wide elution band of coextractants, which may result in lower recoveries.

Instrumental analysis and quantitation The determination of endocrine-disrupting pesticides in foodstuffs requires the use of highly sensitive and selective techniques due to the trace levels at which they are frequently found and the complexity of food matrices.

92

Analysis of Endocrine Disrupting Compounds in Food

For these reasons, the use of chromatographic techniques, coupled with mass-spectrometric detection, is usually the choice of testing methods. The sample preparation procedure should be considered, together with the chromatographic analysis and detection, because these stages are clearly connected. Less specific detection methods must be accompanied by dedicated sample treatment, including cleanup stages. However, if high specific determination methods (e.g., MS/MS with a triple quadrupole or GCxGC) are used, the need for extensive cleanup procedure during sample preparation is substantially reduced. Due to the chemical properties of the organochlorine ED pesticides, gas chromatography (GC) is the separation technique of choice for the determination of these contaminants in food; however, for the most polar organochlorine ED pesticides, such as the herbicides, and some fungicides, the use of liquid chromatography (LC) has rapidly grown in the last few years. The detection systems commonly used in separation techniques are MS, MS/MS, and other traditional nonselective detectors (e.g., electron-capture detector in GC and UV, diode array detection in LC). Gas chromatography-mass spectrometry To date, the most widely used approach for the analysis of organochlorine ED pesticide residues in food has been gas chromatographymass spectrometry (GC-MS). However, the classical electron-capture detector (ECD) has also been used frequently to analyze organochlorine pesticides. According to the guidelines proposed by DG SANCO (European Commission 2006), these detectors do not provide enough selectivity to confirm the presence of a substance. Despite some unique features of conventional detectors (e.g., extreme sensitivity of ECD in detection of polychlorinated analytes), the use of MS techniques is mandatory in order to obtain

unambiguous identification and confirmation. There are various types of MS analyzers. Today, GC-MS, using quadrupole analyzers, is the most widely used. A quadrupole can be operated in two modes: full scan (of selected mass range, usually a mass-to charge ratio, or m/z, of 50–500) and selected ion monitoring (SIM). In SIM mode, sensitivity is enhanced by monitoring only a few ions of interest, but unfortunately spectral information is sacrificed. Various ionization techniques are also possible in GC-MS to provide, in particular cases, higher degrees of information and lower limits of detection (LODs); nevertheless, in the case of multiresidue analysis by GC-MS, electron ionization (EI) is commonly preferred. Chemical ionization (CI), positive chemical ionization (PCI), and negative chemical ionization (NCI), are softer ionization techniques and tend to give lower LODs, depending on the compound, but it is not as widely applicable in multiclass methods and does not provide as much structural information about the analyte as does EI. NCI is highly selective and allows one to obtain lower LODs of compounds that are prone to efficient electron capture, such as organochlorine pesticides. However, under certain circumstances simple MS detectors such as a single quadrupole may fail to detect residues due to high chemical noise overlapped by abundant matrix interference (this may be the case when low, unspecific ions m/z are yielded from an analyte). Either GCxGC separation or the use of MS/MS may provide improved performance. The GC-MS/MS analysis is more accurate than GC-MS; the increased selectivity of MS/ MS techniques reduces the influence of the matrix and also improves the sensitivity compared to both the SIM and full-scan modes. However, it requires careful optimization of several MS/MS parameters of all analytes involved, which is a real drawback of this mode. In general, the main MS/MS instruments are triple quadrupole (QqQ) working

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

in selected reaction monitoring (SRM) and ion-trap (IT). Quadrupole and IT mass spectrometers are commonly used for targeted analysis. The need to select ions (SIM) or MS/MS transitions limits the number of compounds that can be analyzed. The mass spectrometers can be operated in scan mode in the screening or as an untargeted approach to overcome the restrictions encountered with target analysis, although the reduction in acquisition speed, poor response, and interference limit the suitability of such an approach. Time-offlight mass spectrometry (TOF-MS) should overcome many of the limitations and allow coverage of a much larger number of compounds because TOF mass spectrometers provide high performance across the full mass range. TOF-MS provides two complementary approaches (Cajka et al. 2008): (1) instruments that feature unit mass resolution at a high acquisition speed, which predetermines their use as detectors, coupled to fast GC or comprehensive GCxGC and (2) instruments with a moderate acquisition speed, but having high mass resolution, which allows a greater ability to resolve the analytes from the matrix components. Additionally, mass measurement accuracy (<5 ppm) permits estimation of the elemental composition of the detected ions. The unique feature of both TOF-MS techniques is simultaneous sampling and analysis of all ions across the whole mass range (unlike scanning instruments). This permits full-spectrum sensitivity comparable to the SIM mode of a quadrupole instrument. In recent years, application of GC–TOF-MS (both high-resolution and high-speed instruments) has been demonstrated as a powerful and highly effective analytical tool in analysis of food contaminants (e.g., pesticide residues, PCBs, PBDEs, PCDDs, PAHs, etc.), demonstrating great potential of this technique, not only for quantification of target analytes but also for identification of nontarget compounds in complex matrices.

93

Liquid chromatography-mass spectrometry For the most polar organochlorine ED pesticides, such as herbicides and some fungicides, LC coupled to MS (LC-MS) has experienced expanded use in the field of food analysis. In the past, most LC-based methods used common UV-vis and diode-array (DAD) detectors. They are insufficiently selective and sensitive because of the variety and complexity of food and the small amounts of residues present. However, these classical detectors are still applied in some studies even now. The LC-MS analysis of ED pesticides is carried out using atmospheric pressure ionization (API) interfaces, namely electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Both ionization modes complement one another in polarity, molecular mass of analytes, and chromatography conditions. ESI is a very soft ionization technique, especially well suited for ionic compounds and compounds with a high molecular weight. APCI is better suited for nonionic compounds of moderated molecular weight. ESI is more frequently used than APCI because it generally provides better sensitivity for most of the ED pesticides studied in this chapter. Also, a promising new ionization source, atmospheric pressure photoionization (APPI), was introduced just a few years ago. It is an interesting addition to API techniques for increasing the range of LC-MS applications to less polar compounds but is not yet commonly used. Evidently, acid compounds are ionized in negative mode instead of basic compounds, which are ionized in positive mode. The LC-MS quadrupole instrument has been the most commonly used in laboratories until recently because it is relatively inexpensive, rugged yet particularly sensitive in SIM mode, and ideal for trace target applications. However, API techniques usually generate unfragmented spectra. If enhanced collision-

94

Analysis of Endocrine Disrupting Compounds in Food

induced dissociation (CID) is applied, often coeluting compounds form ions of the same m/z values as the analyte, and the resulting multiple component spectrum is virtually useless. More recently, tandem MS (MS/MS) has become a powerful tool for residue analysis in a variety of complex matrices due to its inherent advantages: selectivity and sensitivity are notably improved, the sample pretreatment steps can be minimized, and reliable quantitation and confirmation can be easily achieved at the low concentration levels required. Among the analyzers capable of MS/MS, triple–quadrupole (QqQ) working in selection reaction monitoring (SRM) is the technique most appropriate for target analysis in food because, under these conditions, high sensitivity and selectivity is achieved. Iontrap (IT) is also a commonly used analyzer in food analysis. More recently, TOF-MS and linear traps (LITs), as well as their combination with quadrupoles to attain tandem mass spectrometry QqTOF and quadrupole linear ion trap (QLIT), are gaining acceptance, but their use is not yet general. LC-TOF-MS instruments offer attractive features for the analysis of ED pesticides in complex matrices, such as high mass resolution, mass accuracy, and full-scan spectrum acquisition. TOF analyzers offer improved selectivity due to the high resolution power, enabling the measurement of accurate masses of ions with mass accuracy better than 3 ppm (Lacorte and Fernandez-Alba 2006). Accurate mass analysis of ions usually yields the elemental composition of parent and fragment ions, which are used for unambiguous identification and/or confirmation of a target species or to identify unknown or unexpected compounds. In addition, a consequence of the recording of full-scan spectra with high sensitivity is that it is possible to review and to look for additional compounds not included in the initial analysis. One of the main drawbacks associated with the TOF instrument is that it is not possible to isolate a precursor ion

to obtain clean MS/MS spectra, and fragmentation can be enhanced only if higher fragmenter voltages are applied. In some cases, no fragment ions are obtained with adequate sensitivity. Bearing in mind that this analyzer cannot provide structural information from fragmentation for some compounds, hybrid instruments such as QTOF have been introduced. QTOF can work in single MS as well as MS/ MS operation modes, making this technique a very attractive tool given the combination of high sensitivity, high resolution, and high mass accuracy for both precursor and product ions. QLIT’s unique feature is that the same mass analyzer Q3 can be run in two different modes, retaining the classical triplequadrupole scan functions, such as MRM, product ion, neutral loss, and precursor ion, while providing access to sensitive IT experiments. The very fast duty cycle of QLIT provides a superior sensitivity over that of traditional QqQ and IT and allows one to record product ion scan spectra for confirmation purposes without compromising signalto-noise (S/N) ratio. Although applications in food analysis are still scarce, a few recent papers reported on the application of a hybrid QLIT for trace level determination of EDCs, such as pesticides or PCBs (Payá et al. 2007). Matrix effects and quantitation One of the main drawbacks of LC-MS is that the ionization source is highly susceptible to coextracted matrix components, particularly when studying complex samples such as food. The matrix effect typically results in the suppression or, less frequently, the enhancement of the analyte signal in a sample as compared to a pure standard solution. This phenomenon represents a main source of error in quantitative analysis, affecting many aspects of the method’s performance, such as detection capability, repeatability, or accuracy. ESI interface is more susceptible than APCI to

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

matrix signal-suppression effects, as has been widely discussed (Blasco and Pico 2007). Problems caused by matrix components also represent a major problem in GC-MS. In gas chromatography, the commonly encountered problem is that the sample matrix can cause an enhancement in the observed chromatographic response for analyte residues in matrix extract compared to the same concentration in a matrix-free solution; this phenomenon is called matrix-induced chromatographic response enhancement. The problem originates from the irreversible adsorption of certain sample components on active sites in the GC system, especially in the injection liner. When a matrix-free standard solution is injected into a GC system, active sites are available for analyte absorption. This results in a reduced transfer of analytes into the chromatographic column due to their retention in the injector. The blocking of active sites on the liner by coextracted matrix components will, however, improve the transfer of analytes from the injection port to the column. This phenomenon represents a source of error in quantitative analysis. Matrix interferences are unresolved questions as yet. There are several approaches to reduction of matrix effects in quantitative analysis. Many of them may compensate for matrix effects and result in a quantitatively accurate result, but they cannot avoid the loss of sensitivity that is accompanied by signal suppression and the variability in method sensitivity that may occur between series of samples. This can only be achieved by improving the sample preparation and purification in order to limit the presence of interfering compounds in the final extract. Although this would in principle be the best approach, it may be difficult to achieve, especially in multiresidue analysis, where a variety of interferences may be involved. Other measures to reduce matrix effects in LC-MS include a change to another ionization method, a change in the mobile-phase composition, and/or the use of adequate stan-

95

dardization. In GC-MS, significant elimination of adsorption of analytes is achieved using injection techniques with shorter residence time of the sample stream in the injector, as in the case of PTV injection. A new trend is to add analyte protectants to sample extract. They protect coinjected analytes against degradation, adsorption, or both in GC systems. Their application in practice, however, requires still further research. If matrix suppression/enhancement phenomena cannot be circumvented, appropriate calibration techniques might be used to compensate for matrix effects. This is the most widely accepted and widely used approach in the literature. The best option for a reduction of matrix effects is to perform an appropriate quantification using isotopically labeled internal standards, which are not different in their physicochemical behavior from a corresponding compound, except for their molecular weight. Their use is expensive, especially in a multicomponent analysis where a separate internal standard for each analyte is required. This method is frequently used when only one analyte, or few of them, must be determined. When a large number of target analytes are simultaneously analyzed, alternative methods are used. Quantitation by standard addition method, where the sample extract is spiked with the analytes detected, is one possibility for correcting matrix effects. Nevertheless, this laborious method is not very convenient when a high number of samples need to be analyzed, and it is therefore not frequently used in food analysis. The most widely used approach in MRMs is matrix-matched standard calibration. However, for its application, blank matrices must be available, and this is an inconvenience when a large number of samples with different matrices have to be analyzed. External calibration seems to be the least suitable method, but it is currently used for the analysis of matrices, such as fruits and vegetables, although extensive cleanup procedures must be applied to obtain sufficiently

96

Analysis of Endocrine Disrupting Compounds in Food

clean extracts. If analyte responses in standard solutions and extracts are in agreement, this method can be used.

Analysis of organochlorine endocrine-disrupter pesticides in vegetables, fruits, and cereals Matrices under study can be classified into two main groups: vegetables, fruits, and cereals and products of animal origin. Very different commodities are included within

these groups, and general trends are complicated to establish. In general, in pesticide analysis in crops we can distinguish between three types of matrices: high water content (most of the fruits and vegetables), high oil content (vegetable oil, olives, avocado), and high protein content or high starch content (cereals, potato, carrot, rice). In all cases, it is often necessary in cleanup stages to remove undesirable components of the matrix such as pigments (chlorophyll, carotenoids) and triacylglycerides, lecitine,

Table 4.2. Overview of representative analytical techniques used for the determination of organochlorine endocrine-disrupter pesticides in vegetables, fruits and cereals. Commodity Categories

Vegetables, Fruits, and Cereals Sample Matrix

Preparation Method Pretreatment

Extraction Technical (Sorbent) Elution Solvents

Cleanup/ Derivatization

Orange, cucumber, lemon

Vinclozolin, prochloraz, iprodione

Freeze before comminution

QuEChERS Acetonitrile

DSPE (PSA and anhydrous MgSO4)

Banana, grapefruit

Acetochlor, aldrin, atrazine, pp′DDT, vinclozolin, alachlor Alachlor, atrazine, linuron,diazinon,

Comminuted and homogenized

Solvent extraction Acetonitrile

Comminuted and homogenized

QuEChERS Acetonitrile

Chlordane, lindane, DDT, vinclozolin, dicofol

Comminuted and homogenized

Lindane, vinclozolin, alachlor, triadimefon

Comminuted and homogenized

Solvent extraction Acetonecyclohexane/ ethyl acetate QuEChERS Acetonitrile

SPE (GCB/PSA) Acetonitrile/ toluene DSPE (PSA and anhydrous MgSO4) GPC Bio-Beads SX-3 Ethyl acetate/ cyclohexane DSPE (PSA and anhydrous MgSO4)

BHC, heptachlor, aldrin, DDT, endosulfan alpha, endosulfan beta Acetochlor, alachlor, atrazine, linuron

Comminuted and homogenized

Solvent extraction Ethanol

SPE (Florisil) Toluene

Comminuted and homogenized

QuEChERS Acetonitrile

DSP (PSA and anhydrous MgSO4)

Tomato, pear, orange

High Water Content

Compounds

Apple, pear, lettuce, tomato, cabbage, onion, leek, orange, grapefruit Aubergine, cucumber, banana, tangerine, orange, melon, tomato Lettuce, onion, spinach, leaf mustard, celery, cabbage Apple, pepper, kiwi, lettuce, tomato, strawberry, pear cucumber

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

etc. Probably the most complex matrices are those with high fat content because it is quite complex to extract the studied compounds without coextraction of lipids, which usually are difficult to remove from the extract and may harm the detection system.

Sample preparation An overview of the most recent methodologies reported to analyze organochlorine

97

endocrine-disrupter pesticides in vegetables, fruits, and cereals is shown in Table 4.2. As can be observed in the table, cutting the sample into small pieces and mixing it in a blender, mixer, or in an Ultra Turrax is the pretreatment usually used in fruits and vegetables prior to extraction. Losses of some pesticides have been reported when samples of fruits and vegetables are comminuted at ambient temperature (Fussell et al. 2002). Some authors minimize the loss of pesticides at the sample pretreatment stage by freezing

Vegetables, Fruits, and Cereals Analytical Method Detection Mode

LC or GC Column Mobile Phase

LC-QLIT-MS/MS ESI (+), MRM GC-QqQ-MS/MS Use of analyte protectans

LC-QLIT-MS/MS: C18 Water (5 mM ammonium formate)/methanol (5 Mm ammonium formate ) GC-QqQ-MS/MS: DB-5MS DB-5MS

LODs (μg/Kg or μg/L*)

LOQs (μg/Kg or μg/L*)

Recovery (%)

Reference

86–120 i,e

Payá et al. 2007

0.3–18.8

70–120 e

Okihashi et al. 2007

C18 Water (0.1% formic acid)/acetonitrile HP-5MS

≤5

70–120 e,s

Kmellar et al. 2008

GC-Q-MS (EI), full scan

HP-5MSi

≤20

GC-ECD GC-FID

HP-5 Ultra-2 capillary column

LC-TOF-MS LC-QqQ-MS/MS ESI (+), MRM

LC-TOF-MS: C18 Water/acetonitrile (0.1% formic acid)/ acetonitrile/water (0.1% formic acid) LC-QqQ-MS/MS: Water (0.1% formic acid)/acetonitrile

GC-QqQ-MS/MS (EI), MRM LC-QqQ-MS/MS ESI (+) GC-Q-MS (EI), SIM

Knezevic and Sedar 2009 ≤20

80–110 e,s

Mezcua et al. 2009

20–50

65–95

Wan et al. 1994

5–10

e

Mezcua et al. 2008

(continued)

Table 4.2. Overview of representative analytical techniques used for the determination of organochlorine endocrine-disrupter pesticides in vegetables, fruits and cereals. (cont.) Commodity Categories

Vegetables, Fruits, and Cereals Sample Matrix

Apples, pear, apricot Grapes

Tomato, cucumber, pepper High Water Content

Preparation Method Pretreatment

Apple

Grapes, apples, peaches, apricots, oranges… Lemon, tomato, apple, grape, banana, mango, onion, cucumber Cucumber, cabbage, cole, pepper Lettuce, cabbage, leek Lettuce, chard, spinach

98

Compounds

Diuron, linuron and metabolites DCPMU, DCPU and 3,4-DCA Atrazine ….

Extraction

Comminuted and homogenized

Technical (Sorbent) Elution Solvents MSPD (Florisil) Methanol

Comminuted and homogenized Comminuted and homogenized

UA/MSPD (C8) Ethyl acetate Solvent extraction Ethyl acetate

Comminuted and homogenized

Solvent extraction Ethyl acetate

Atrazine, chlordane, DDT, vinclozolin, HCH, aldrin, dieldrin, heptachlor… BHC, heptachlor, aldrin, endosulfan, dieldrin, endrin Chlordane: αChlordane, β-chlordane and oxychlordane α-HCH, β- HCH, γ-HCH and δ-HCH

Comminuted and homogenized

Solvent extraction Petroleum ether: acetone

Comminuted and homogenized

BHC, DDT, endosulfan

Comminuted and homogenized

MSPD (Florisil) n-Hexane:ethyl acetate Solvent extraction Acetonedichloromethane

Atrazine, diazinon, simazine, vinclozolin,nitrofen, prochloraz Vinclozolin, iprodione, triadimefon

Comminuted

QuEChERS Acetonitrile

Comminuted

Solvent extraction Acetonitrile in ultrasound bath

Cleanup/ Derivatization

DSPE (PSA)

Florisil column Cyclohexane-ethyl acetate GPC-column chromatography Florisil

SPE (GCB in combination with acidic aluminum oxide) DSPE (PSA, GCB, and anhydrous MgSO4) SPE (GCB/PSA)

Vegetables, Fruits, and Cereals Analytical Method Detection Mode

LC or GC Column Mobile Phase

LODs (μg/Kg or μg/L*)

HPLC-UV-DAD

C18 Water/Acetonitrile

1.8–5.0

GC-Q- MS (EI), full scan GCxGC-TOF-MS GC-TOF-MS ESI (+)

BPX5

LOQs (μg/Kg or μg/L*) 6.9–20.9

Recovery (%)

Reference

54–84 i

Boti et al. 2009

1–205

71–139

RTX-5MS TR-50MS

GCxGC-TOFMS: 0.2–3 GC-TOF-MS: 2–19

67–109 e

Ramos et al. 2008 Banerjee et al. 2008

GC-ECD

Phenomenex zebron zb 1701

0.92–2.92*

GC-ECD two ECD

DB-5 DB-17

GC-ECD

Elite 35

GC-IT-MS/MS full scan

DB-5MS

GC-QqQ-MS/MS (EI), MRM

DB-5MS

GC-IT-MS/MS full scan, SIM Use of analyte protectans

SPB-5

1.87–11.2*

62–108 e

YenisoyKarakas 2006

0.002–0.05

101–103 i

Janouskova et al. 2005

3–6

93–103 e

Abhilash et al. 2009

20–100

64–36 e

Tao et al. 2009

5–70

70–120 i, e

Walorczyk 2008

1–34

79–133 i

GonzálezRodríguez et al. 2008

1–16

(continued)

99

Table 4.2. Overview of representative analytical techniques used for the determination of organochlorine endocrine-disrupter pesticides in vegetables, fruits and cereals. (cont.) Commodity Categories

Vegetables, Fruits, and Cereals Sample Matrix

High Water Content

Preparation Method Pretreatment

Extraction Technical (Sorbent) Elution Solvents QuEChERS Acetonitrile

Cleanup/ Derivatization DSPE (PSA and anhydrous MgSO4)

Solvent extraction Acetone:ethyl acetate:hexane in ultrasonic water bath Solvent extraction Acetonitrilepetroleum ether Solvent extraction Acetone

SPE (RPC18) 5% acetone in hexane

MSPD (C18-bonded silica) N-hexane: ethyl acetate Solvent extraction Methanol:water 0.1% acetic acid

Quartz distilled water

Orange, lettuce

Linuron, endosulfan

Comminuted and homogenized

Cucumber, spring onion, strawberry, papaya

Endosulfan

Comminuted and homogenized

Cucumber

HCB, heptachlor, aldrin, endrin, DDT and dieldrin Diazinon, linuron, prochloraz, diuron

Comminuted and homogenized

Pyrethroid pesticides

Comminuted and homogenized

Tomato, lemon

Atrazine, diuron, linuron

Comminuted and homogenized

Apple, celery, lemon, pear

Simazine, atrazine, lindane, alachlor, aldrin, heptachlor epoxide, chlordane Simazine, atrazine, alachlor, acetochlor, and nitrofen

Comminuted and homogenized

Solvent extraction Methylene chloride

Microwave inactivation of the enzyme alliinase preventing formation of sulfurcontaining compounds in onion: Heated in a microwave oven for 30 s Comminuted and homogenized

Solvent extraction Acetonitrile

SPE (Florisil) Acetone:n-hexane

Solvent extraction Distilled water: light petroleum

SPE (C18) Ethyl acetate

Lettuce, orange, apple, cabbage, grape Orange, apple, pear, grape

Onion

Onion, tomato, lettuce, spinach, green pepper, aubergine, cucumber

100

Compounds

Lindane, HCB, vinclozolin, heptachlor, aldrin, dicofol, chlordane, iprodione, endrin

Comminuted and homogenized

SPE (Florisil) Diethyl ether/ petroleum ether

SPE (Oasis HLB) Methanol:(MTB 0.1%) acetic acid

Vegetables, Fruits, and Cereals Analytical Method Detection Mode

LC or GC Column Mobile Phase

GC-IT-MS/MS: (EI), full scan mode LC-QqQ-MS/MS: ESI (+)

CP-Sil 8-MS C18 MeOH: water (5mM HCOOH)/ MeOH:water (5 mM HCOOH) SGE BPX5 HP-5MS

GC-ECD GC-Q-MS: SIM

LODs (μg/Kg or μg/L*)

GC-ECD: 5– 10 GC/MS (Q): 10– 50

LOQs (μg/Kg or μg/L*)

Recovery (%)

Reference

10

90–110 i, e

Lehotay et al. 2005

GC-ECD: 10–20

86–102 i

Lal et al. 2008

GC-ECD

HP-101

3–43

76–102 e

Mansour et al. 2009

LC-QqQ-MS/MS (Z-spray) positive mode, MRM GC-Q-MS (EI), SIM

C18 MeOH/water (5mM ammonium formate) HP-5MS

10

70–110

Hiemstra and Kok 2007

31–118

Kristenson et al. 2001

LC-QqQ-MS/MS ESI (+)

C18 Methanol (0.01% HCOOH))/water (0.01% HCOOH) RTX-5MS

70–110 e

Hernández et al. 2006

1–5

50–180 i,e

Schachterle and Feigel 1996

GC-Q-MS (EI), SIM

DB-5MS

1.5–3

69–105 e

Zhang et al. 2008

GC-ECD

Fused-silica capillary column

0.04–10

64–112 i

Columé et al. 1999

GC-IT-MS/MS

4–90

10

3–15

(continued)

101

Table 4.2. Overview of representative analytical techniques used for the determination of organochlorine endocrine-disrupter pesticides in vegetables, fruits and cereals. (cont.)

High Water Content

Commodity Categories

Vegetables, Fruits, and Cereals Sample Matrix

Extraction

Grape, lettuce, orange

Aldrin, chlordane, DDT, dieldrin, endosulfan, endrin, lindane, heptachlor

Comminuted and homogenized

Technical (Sorbent) Elution Solvents Solvent extraction Ethyl acetate

Apple

HCB, dimethoate, lindane, heptachlor, vinclozolin, endosulfan

Comminuted and homogenized

QuEChERS Acetonitrile

DSPE (PSA and anhydrous MgSO4)

Red grape

Vinclozolin, prochloraz, iprodione

Freeze before comminution

QuEChERS Acetonitrile

DSPE (PSA and anhydrous MgSO4)

Vegetable juices (carrot, grape, tomato) Orange juice

Simazine, lindane, alachlor

MSPD (Florisil)

Sonication Ethyl acetate

Diuron, linuron and metabolites (DCPMU, DCPU and 3,4-DCA) Atrazine, lindane, alachlor, aldrin, endosulfan, triadimefon

Comminuted and homogenized

MSPD (Florisil) Methanol

Methanol addition

SPE (C18) Hexane-ethyl acetate

Apple

High Oil Content

Preparation Method Pretreatment

Vegetable juices (carrot, peach, grape, orange, pineapple, and apple) Tomato juice

102

Compounds

Endosulfan-alpha, endosulfan-beta and endosulfan sulfate HCB, lindane, heptachlor, vinclozolin, endosulfan

Cleanup/ Derivatization

MSPD (Florisil) Ethyl acetate Comminuted and homogenized

QuEChERS Acetonitrile

DSPE (PSA and anhydrous MgSO4) SPE (Oasis HLB SPE cartridges) Methanol: (MTBE 0.1%) acetic acid Florisil

Avocado

Atrazine, diuron, linuron

Comminuted and homogenized

Solvent extraction Methanol:water 0.1% acetic acid

Olive oil

Lindane, vinclozolin, alachlor, triadimefon

Comminuted and homogenized

LLE Petroleum ether/ acetonitrile MSPD (Aminopropil) Acetonitrile

Vegetables, Fruits, and Cereals Analytical Method Detection Mode

GC-QqQ-MS/MS: (EI) LC-QLIT-MS/MS: ESI (+) and ESI (−) GC-Q-MS: NCI, methane reagent gas (EI), SIM

VF-5 C18 Methanol/10 mM ammonium formate (ph:4) HP-5MS

LC-QLIT-MS/MS ESI (+), MRM

LC-QLIT-MS/MS: DB-5MS Water (5 mM ammonium formate/methanol) (5 Mm ammonium formate ) ZB-5MS

GC-MS (EI), SIM

LODs (μg/Kg or μg/L*)

LC or GC Column Mobile Phase

NCI: 0.00564– 0.935* EI: 0.10–3.12*

0,1–1,6

LOQs (μg/Kg or μg/L*)

Recovery (%)

Reference

70–120 e

Pihlstrom et al. 2007

e

Huskovaa et al. 2009

84–119 i,e

Payá et al. 2007

0,3–5,3

82–104 i

Albero et al. 2004

5–8.8

57–96 i

Boti et al. 2009

NCI: 0.00632– 3.114* EI: 0.30–10,4*

HPLC-UV-DAD

Discovery C18Water/ acetonitrile

GC-Q- MS (EI), SIM

ZB-5MS

0.1–4.6*

0.3–15.2*

96.5–101 i

Albero et al. 2005

GC-ECD GC-Q-MS (EI), SIM GC-Q-MS NCI, methane reagent gas (EI), SIM

HP-1 ZB-5MS

1

3

81– 100,6 e

Albero et al. 2003

HP-5MS

NCI: 0.00564– 0.935* EI: 0.10–3.12*

NCI: 0.00632– 3.114* EI: 0.30–10,4*

e

Huskovaa et al. 2009

LC-QqQ-MS/MS ESI (+)

C18 Methanol (0.01% HCOOH)/water (0.01% HCOOH)

10

70–110 e

Hernández et al. 2006

GC-Q-MS (EI), full scan Retention time locked Backflush

HP-5MSi

≥20

80–110 e, s

Mezcua et al. 2009

≥20

(continued)

103

Table 4.2. Overview of representative analytical techniques used for the determination of organochlorine endocrine-disrupter pesticides in vegetables, fruits and cereals. (cont.)

High Protein Content or High Starch Content

High Oil Content

Commodity Categories

Vegetables, Fruits, and Cereals

104

Sample Matrix

Compounds

Preparation Method Pretreatment

Olive oil

Atrazine, simazine, endosulfan

Vegetable oil

Hexaclorobenzene, endrin, aldrin, chlordane, heptachlor

Olive oil

Simazine, atrazine, diuron

Olives

Simazine, atrazine, diuron

Olive oil

Endosulfan I, II, sulfate, cypermethrin

Olives

Vegetable oil

Endosulfan I, II, sulfate, methylcypermethrin Cypermethrin, atrazine

Olive oil

Lindane,dieldrin

Wheat flour

Vinclozolin, prochloraz, iprodione

Carrot Potato

Potato, carrot

Extraction Technical (Sorbent) Elution Solvents LLE Hexane/acetonitrile

Cleanup/ Derivatization SPE: ENVI-Carb SPE cartridgeDiol SPE cartridge GPC Ethyl acetate: cyclohexane

LLE Petroleum ether: AcN MSPD (Aminopropyl) MSPD (Aminopropyl)

SPE (Florisil) Acetonitrile

GPC mobile phase addition (ethyl acetatecyclohexane)

Homogenized

SPE (Florisil) Acetonitrile

LLE Petroleum ether:AcN and MSPD (Aminopropyl) MSPD (Aminopropyl)

SPE (Florisil) Acetonitrile

SFE

SPE (Florisil)

LLE AcN/n-hexane

GPC Ethyl acetateciclohexane

Employed directly for extraction Cold water addition

QuEChERS Acetonitrile

DSPE (PSA and anhydrous MgSO4)

Acetochlor, aldrin, atrazine, pp′ DDT, vinclozolin, alachlor Chlordane, lindane, DDT, vinclozolin, dicofol

Comminuted and homogenized

Solvent extraction Acetonitrile

Comminuted and homogenized

Lindane, vinclozolin, alachlor, triadimefon

Comminuted and homogenized

Solvent extraction Acetonecyclohexane/ ethyl acetate QuEChERS Acetonitrile

SPE (GCB/PSA) Acetonitrile/ toluene GPC Bio-Beads SX-3 Ethyl acetate/ cyclohexane DSPE (PSA and anhydrous MgSO4)

Homogenized

SPE (Florisil) Acetonitrile

Vegetables, Fruits, and Cereals Analytical Method Detection Mode

LC or GC Column Mobile Phase

GC-ECD GC-NPD GC/MS: (EI),SIM GC-QqQ-MS/MS (EI), MRM

Zebron ZB-5 Zebron ZB-1

LC-IT-MS/MS (ESI+)

XDB-C18 AcN/water (0.1%formic acid)

LC-IT-MS/MS (ESI+)

XDB-C18 AcN/water (0.1%formic acid) ZB-5MS

GC-Q-MS (CI−), SIM

ZB-5MS

GC-ELCD (organochlorine) GC-ECD GC-TSD GC-IT-MS/MS: (EI)

DB-1 8 (organochlorine)

LC-QLIT-MS/MS ESI(+), MRM GC-QqQ-MS/MS Use of analyte protectans

LC-QLIT-MS/ MS:DB-5MS Water (5 mM ammonium formate/methanol (5 Mm ammonium formate ) GC-QqQ-MS/MS:C18 DB-5MS

GC-Q-MS (CI−), SIM

GC-QqQ-MS/MS MRM

VF-5MS

CP-SIL 5CB CP-SIL 8CB CP-SIL 5CB

GC-Q-MS (EI), SIM

HP-5MS

GC-Q-MS (EI), full scan Retention time locked Backflush

HP-5MSi

LODs (μg/Kg or μg/L*) 0.8–13.1 (GC/ ECD) 0.4–14.5 (GC/NPD) 0.1–2*

LOQs (μg/Kg or μg/L*)

Reference

88.6–106.7i (GC/ECD) 70.9–106.4i (GC/NPD) 63–116 i,e

Amvrazi and Albains 2009

0.1–2*

83–103 e

Ferrer et al. 2005

0.4–4

81–96 e

Ferrer et al. 2005

3–60*

73–103 e

Ferrer et al. 2005

8–80*

95–113 e

Ferrer et al. 2005

0–118

Hopper 1999

94–124 82–100 89–105 e,i

Guardia-Rubio et al. 2006

69–112 i,e

Payá et al. 2007

0.5–10* 2–10* 0.2–10*

2.6–43.3 (GC/ECD) 1.6–47.8 (GC/NPD)

Recovery (%)

2–10* 5–20* 0.5–20*

0.3–18.8

Patel et al. 2005

70–120 e Knezevic and Sedar 2009

≥20

≥20

80–110e, s

Mezcua et al. 2009

(continued)

105

Table 4.2. Overview of representative analytical techniques used for the determination of organochlorine endocrine-disrupter pesticides in vegetables, fruits and cereals. (cont.) Commodity Categories

Vegetables, Fruits, and Cereals Sample Matrix

Preparation Method Pretreatment

Cereal/wheat flour

Potato, carrot

Rice

Leaf, stem, wheat green gram, medicinal plants and root Rice High Protein or High Starch Content

Compounds

Barley

Diuron, linuron and metabolites DCPMU, DCPU, and 3,4-DCA Diuron, linuron and metabolites DCPMU, DCPU, and 3,4-DCA BHC, vinclozolin, lindane, DDT, triadimefon

Comminuted and homogenized Comminuted and homogenized

MSPD (Florisil) Methanol

Sample saturated sodium chloride

alpha-HCH, betaHCH,γ-HCH, and δ-HCH

Dried at 35°C for 24 h, powdered and sieved Add water before extracting

Solvent extraction Dichloromethane Ultrasonic extraction MSPD (Florisil) n-hexane:ethyl acetate QuEChERS Acetonitrile

Acetochlor, alachlor, BHC, endrin, heptachlor, iprodione, DDT Linuron and 2,4-D

Add water before extracting

QuEChERS Acetonitrile Mini-Luke Acetone/ dichloromethane/ light petroleum Ethyl acetate extraction Acetone extraction Solvent extraction Acetone

Cleanup/ Derivatization

SPE (Florisil) Hexane and acetone Alumina

DSPE (PSA, GCB, and anhydrous MgSO4) DSPE (PSA and anhydrous MgSO4) GPC (only for the AcEt extraction)

Wheat flour

Linuron, prochloraz, diuron

Carrot

Aldrin, chlordane, DDT, dieldrin, endosulfan, endrin, lindane, heptachlor

Comminuted and homogenized

Solvent extraction Ethyl acetate

Carrot

Simazine, atrazine, lindane, alachlor, aldrin, heptachlor epoxide, chlordane Lindane, HCB, vinclozolin, heptachlor, aldrin, dicofol, chlordane, iprodione, endrin HCH, DDT, heptachlor, epoxide, aldrin, dieldrin, and endrin

Comminuted and homogenized

Solvent extraction Methylene chloride

Comminuted and homogenized

Solvent extraction Distilled water: light petroleum

SPE (C18) Ethyl acetate

Dried at room temperature Removed bran Samples dried and powdered

Solvent extraction Petroleum ether Ultrasonic water bath at room temperature

SPE (Florisil) Ethyl acetate and petroleum ether

Carrot, potato

Rice

i, Internal calibration; e, external calibration; s, with surrogate standard.

106

Extraction Technical (Sorbent) Elution Solvents MSPD (Florisil) Methanol

Vegetables, Fruits, and Cereals Analytical Method Detection Mode

LC or GC Column Mobile Phase

LODs (μg/Kg or μg/L*)

LOQs (μg/Kg or μg/L*)

Recovery (%)

Reference

HPLC-UV-DAD

C18 Water/Acetonitrile

1.8–5.0

5.6–17.1

57–92 i

Boti et al. 2009

HPLC-UV-DAD

Discovery C18 water/Acetonitrile

1.8–5.0

5.3–16.1

49–96 i

Boti et al. 2009

GC-Q-MS SIM

HP-5MS

0.049–9.7

75–120 e

Pengyan et al. 2006

GC-ECD

Elite 35

3–6

93–103 e

Abhilash et al. 2009

GC-Q-MS SIM

DB-5MS

75–113 e

Nguyen et al. 2008

GC-TOF-MS LC-QqQ-MS/MS ESI(+) and ESI(−), SRM

RTX-CL C18 Water:MeOH (5 mM HCOOH)/ MeOH:water (5mM HCOOH)

60–108 i,e (QuEChERS) 40–95 i, e (EtAc) 60–141i,e (mini-Luke) 61–105 i,e (AcEt)

Díez et al. 2006

LC-QqQ-MS/MS (Z-spray) positive mode GC-QqQ-MS/MS: (EI) LC-QLIT-MS/MS: ESI (+) and ESI (−) GC-IT-MS/MS

C18 MeOH/water (mM ammonium formate) VF-5 C18 Methanol/1mM ammonium formate (pH:4) RTX-5MS

70–110

Hiemstra and Kok 2007

70–120 e

Pihlstrom et al. 2007

1–5

50–180 i, e

Schachterle and Feigel 1996

GC-ECD

Fused-silica capillary column

0.04–10

64–112 i

Columé et al. 1999

Two-dimensional GC-ECD

DB-1701 HP-5MS

0.6–8

81.4–90.4 e

Chen et al. 2007

4–50

0.2–23.2 (QuEChERS, LC-QqQMS)

10

107

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Analysis of Endocrine Disrupting Compounds in Food

the samples and mixing them in the presence of dry ice (cryogenic milling) (Payá et al. 2007). However, special treatments might be required depending on the matrix composition and nature of the analytes. As an example, Zhang et al. (2008) reported an efficient procedure to prevent the formation of sulfurcontaining compounds in onions, which affect the determination of herbicides, by microwave pretreatment of the onion to inactivate the enzyme alliinase. In contrast to the methods developed in the past, the general trend and the most common strategy in the analysis of pesticides in fruits and vegetables over recent years has been the development of multiresidue methods (MRMs), which allow proper control of a large number of different pesticides in a single analysis. Nevertheless, simultaneous analysis of compounds with different physicochemical properties requires a compromise in the selection of experimental conditions for all analytes. Many multiresidue procedures, employing different cleanup techniques and a variety of detection methods, have been reported for the determination of pesticide residues, including the studied compounds, in fruits and vegetables (Table 4.2). In most cases, the extraction method is still based on solvent extraction, methods that have been employed with satisfactory results. The choice of solvent is one of the most important decisions to make in the multiresidue method. The extraction solvents most commonly used in MRMs for the determination of pesticides in fruits and vegetables are acetonitrile (Okihashi et al. 2007; Gónzalez-Rodríguez et al. 2008); ethyl acetate (Yenisoy-Karakas 2006; Banerjee et al. 2008; Pihlstrom et al. 2007); acetone (Hiemstra and Kok 2007); or a combination of acetone, acetonitrile, or water with a nonpolar solvent (Janouskova et al. 2005; Knezevic and Sedar 2009). Acetonitrile extraction methods have been broadly applied since the introduction of the QuEChERS method. The main advantages of

solvent extraction are simplicity and effectiveness, but the large volume of solvents and lack of automation are among the main pitfalls of this widely used method. Most of the solvent extraction–based methods described in the literature for the analysis of the selected EDCs in fruits and vegetables include a cleanup stage. SPE is, so far, the most popular cleanup technique. Although several types of adsorbent are available, early methods in development are centered on the use of Florisil (Yenisoy-Karakas 2006; Mansour et al. 2009; Zhang et al. 2008; Wan et al. 1994; Yenisoy-Karakas 2006); reversed-phase C18 (Lal et al. 2008; Columé et al. 1999); or Oasis HLB (Hernández et al. 2006). Lately, some treatment protocols focus on the use of a combination of two or more commercially available SPE columns for more effective cleanup. An example is the combination of graphitized carbon black (GCB) and primary secondary amine (PSA) columns as dual-layer SPE cartridges (Okihashi et al. 2007; González-Rodríguez et al. 2008). Another effective way in which to use SPE sorbents for removing coextractants from the matrix is by mixing the SPE material with the crude extract: the approach is called dispersive solid-phase extraction (DSPE) (Banerjee et al. 2008) and is the cleanup procedure used in the QuEChERS method. GPC has also been used in pesticide residue cleanup of fruits and vegetables, normally using glass columns packed with divinylbenzene-linked polystyrene gel sorbent (Bio-Beads SX-3) and a mixture of ethyl acetate and cyclohexane as the elution solvents (Knezevic and Sedar 2009). An additional cleanup with Florisil is sometimes included after the GPC step (Janouskova et al. 2005). In sample preparation, there is a trend to shift from laborious traditional methods to new, fast and simple approaches, such us the QuEChERS multiresidue method. This method effectively covers a very wide analyte scope. As can be observed in Table 4.2, in the recently developed multiresidue methods for

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

the analysis of pesticide residue, including the organochlorinated ED pesticides, the QuEChERS method has been widely used (Payá et al. 2007; Kmellar et al. 2008; Mezcua et al. 2008; Walorcyk et al. 2008; Huskovaa et al. 2009). The basic procedure, as noted previously, is based on a single-step acetonitrile extraction and salting out by liquid– liquid partitioning from the water in the sample with MgSO4, followed by a dispersive solid-phase extraction cleanup with PSA. However, some authors have proposed some variants of this method. Recently, Walorczyk and coworkers (2008) have reported a modified QuEChERS method for the analysis of 129 pesticides in green leafy vegetables. The method is based on liquid partitioning with acetonitrile, but the cleanup is performed with GCB and PSA, instead of only PSA. GCB was applied for the purification of the extracts due to its decolorizing properties in the analysis of samples with a high content of pigments, and it was found that an amount between 10 and 15 mg GCB per milliliter of an acetonitrile extract containing 1 g sample was appropriate to remove most pigments while maintaining quantitative recovery of the target pesticides. Matrix solid-phase dispersion (MSPD) is also a relatively recent extraction technique, used for the simultaneous determination of various pollutants from semisolid and solid samples. Recently, MSPD-based methods have been successfully applied for the analysis of organochlorine ED pesticides in fruits and vegetables. Reversed-phase material (C18 and C8) (Kristenson et al. 2001; Ramos et al. 2008) and Florisil (Boti et al. 2007; Abhilash et al. 2009) are the sorbents most commonly applied in MSPD for pesticide analysis in food, and the most commonly used solvent is ethyl acetate (Kristenson et al. 2001; Ramos et al. 2008; Abhilash et al. 2009). Boti et al. (2007) demonstrated the applicability of MSPD in the determination of two organochlorine ED pesticides, linuron and diuron, and their common metabolites, in different

109

food commodities, including fruits and vegetables. They used Florisil as the sorbent material and methanol as the eluting solvent, followed by high-performance liquid chromatography (HPLC)/UV-DAD determination. An extra amount (0.5 g) of Florisil material at the bottom of the MSPD column was used as an additional cleanup phase. The influence of the main factors on the extraction process yield was evaluated using a fractional factorial design in combination with artificial neural networks. Kristenson et al. (2001) developed and validated miniaturized and automated MSPD method using C8-bonded silica as the sorbent and subsequent GC-MS analysis for the trace-level determination of pesticide residues in fruit. Ramos et al. (2008) developed a method based on ultrasonicassisted matrix solid-phase dispersion (UAMSPD) with a sonoreactor for extraction and cleanup of 15 organophosphorus pesticides and 9 triazine (including chlorinated ED herbicides) in fruits prior to GC-MS analysis. Compared with classic MSPD, the proposed method improved the general extraction efficiency, decreased the relative standard deviations, and allowed complete sample treatment within a few minutes. Determination of pesticides in food is often complicated by the presence of different fat content. A distinction should be made when developing methodologies for the sample treatment of fatty vegetable matrices with relatively high fat content (i.e., up to 25% fat) and edible vegetable oils (100% fat composition), the latter being generally more complex. Sample preparation is therefore a crucial step in the analytical procedure because even small amount of lipids can harm columns and detectors; however, analytical determination is less problematic than a sample treatment step. To date, LLE is probably the main strategy used for the sample treatment of organochlorinated ED pesticides in vegetable oils (see Table 4.2). When LLE is used, a cleanup step is necessary because the extracts obtained after this extraction

110

Analysis of Endocrine Disrupting Compounds in Food

method contain certain amounts of fat. For the extraction of vegetable oils, LLE can be used with SPE purification (Amvrazi and Albanis 2009), combined with lowtemperature fat precipitation, or for preliminary partitioning/fractionation before MSPD (Ferrer et al. 2005) or GPC (Guardia-Rubio et al. 2006). GPC is an extensively used technique to analyze pesticide residues in vegetable oil generally after a preliminary LLE, although there are methods based on direct extraction and cleanup of pesticides from oil without a LLE step, using only GPC (Patel et al. 2005). However, to lengthen the life of the columns and chromatographic systems and to obtain cleaner extracts, it is recommended that a partitioning step be performed prior to GPC. Ferrer et al. (2005) proposed a simple sample preparation strategy for the multiresidue determination of pesticides in olives based on MSPD, using aminopropyl as sorbent material and acetonitrile as eluting solvent. The protocol included a cleanup step using a cocolumn packed with Florisil. A similar approach was also proposed by the authors for the same determination on olive oil samples, using an additional LLE with acetonitrile saturated with petroleum ether. Hopper (1999) developed a multiresidue method for the determination of organochlorine and organophosphate pesticides in fatty matrices using an automated supercritical fluid extraction (SFE) and in-line cleanup system, which uses Florisil sorbent. The automated system recovers 86 of 117 nonpolar to moderately polar pesticides from fats. There is scarce literature available on the use of SFE to determine pesticides in fatty vegetable matrices.

Instrumental analysis and quantitation To date, the most widely used technique for the analyses of organochlorinated ED pesticide residues in fruits and vegetables is GC-MS, although multiresidue methods

based on LC-MS/MS are growing in popularity. In GC, most studies use a typical nonpolar column with a phase composition equivalent to 5% phenyl and 95% polydimethyl siloxane. In general, splitless injection was the preferred injection technique. Furthermore, programmed-temperature vaporization (PTV) was also applied. The mass-spectrometric detection system is replacing the formerly used nonselective detection system, but many methods still employ ECD (Mansour et al. 2009; Abhilash et al. 2009; Yenisoy-Karakas 2006; Columé et al. 1999) for the analysis of organochlorine pesticides. The most widely used technique in GC-MS is the quadrupole analyzer in selected ion-monitoring mode (Knezevic and Sedar 2009; Kristenson et al. 2001; Zhang et al. 2008; Ferrer et al. 2005; Nguyen et al. 2008). However, the results, in terms of selectivity, sensitivity, and confirmation degree, improve when tandem mass spectrometry is applied. GC-MS/MS, using either ion trap (González-Rodríguez et al. 2008; Lehotay et al. 2005; Guardia-Rubio et al. 2006; Schachterle and Feigel 1996) or triple quadrupole (Walorczyk 2008; Payá et al. 2007; Pihlstrom et al. 2007; Patel et al. 2005) instruments, has increased in recent years because these techniques enable the unambiguous identification and quantification of a large number of compounds in complex food matrices. The use of SIM and MS/MS mode take time, which limits the number of targeted analytes that can be detected in a given time period. The sensitivity and selectivity of detection is increased, especially in MS/MS mode, but full-scan operation (monitoring the whole spectrum) provides sufficient information for confirmation and can monitor hundreds of analytes, whereas SIM and MS/MS is typically limited to about 100 pesticides in a single chromatogram. In addition, SIM and MS/MS modes rely on only a few ions and are not designed to find compounds unless they are on the target list. With the full-scan

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

mode, identification of nontarget compounds and library-based screening is possible. Very recently, Mezcua et al. (2009) have developed a full-scan GC-Q-MS method to perform large-scale screening of pesticide residues in fruits and vegetables and simultaneous quantification of 95 target compounds in a single run of 21 min. The screening method was performed by using a deconvolution of the full-scan spectrum together with a database containing mass spectra and locked retention times for 927 pesticides and EDCs. With retention time locked (RTL)-GC-MS, the detection selectivity is greatly improved by linking the locked retention time to mass spectral data. This reduces the risks of false positives. The automated mass spectral deconvolution and identification system (AMDIS) used by the authors is a postprocessing software for extracting purified mass spectra from a one-dimensional gas chromatogram and offers the possibility of identifying compounds by both mass spectra and retention time together. In recent years, application of GC–TOFMS (both high-resolution and high-speed instruments) has been demonstrated as a powerful and highly effective analytical tool in the analysis of food and environmental contaminants. A feature of this technique is simultaneous sampling and analysis of all ions across the whole mass range (unlike scanning instruments). This permits fullspectrum sensitivity comparable to selected ion monitoring (SIM) mode of a quadrupole instrument. TOF-MS that features unit mass resolution at high acquisition speed (up to 500 spectra/s) is used as a detector coupled to comprehensive two-dimensional GC (GCxGC). GCxGC–TOF-MS is a powerful separation and identification technique that is suitable for the analysis of complex food samples, which often contain hundreds of GC-amenable compounds. When using GCxGC, the analytes of interest can not only be better separated from each other but, more importantly, can also be separated from

111

matrix compounds, which tend to seriously interfere in 1D-GC–MS procedures. This technique can provide group-type separations. In the latter instance, the ordered structure of the final chromatogram facilitates analyte identification. Consequently, the quality of the TOF-MS mass spectra obtained by GCxGC is much better than those obtained with 1D-GC. Banerjee et al. (2008) have developed and optimized a GCxGC technique for separation of 51 pesticides in grape matrix followed by TOF-MS analysis. This powerful technique facilitated automated library-based screening of the residues at 10 ng/g level. The tendency toward the use of new polar pesticides has prompted the use of LC-MS, which has been accepted as a routine technique for regulatory monitoring purposes in pesticide residue analysis. Used only for the organochlorine pesticides, GC-MS is advantageous as compared with LC-MS, due to the low polarity and lack of ionizable moieties of these apolar compounds by LC-ESI-MS. To achieve better sensitivity and selectivity of target analyte detection, tandem mass spectrometry (MS/MS) is generally the preferred option for quantitation purposes. The use of LC-MS/MS with triple quadrupole (QqQ) instruments in multiple reaction monitoring (MRM) mode is so far the more appropriate technique for target analysis (Kmellar et al. 2008; Hiemstra and Kok 2007; Hernández et al. 2006). Nevertheless, in recent years, TOFMS, Q-TOF, and quadrupole linear ion trap (QLIT) detectors have increased their number of applications. QLIT instruments outperform the conventional ion trap system and deliver the functionality of QqQ, although applications of this technique in food analysis are still scarce. Recently, some papers have reported the application of a hybrid QLIT for trace level determination of EDCs, such as organochlorine pesticides or PCBs (Payá et al. 2007; Pihlstrom et al. 2007). The main advantages of the TOF analyzers are high sensitivity in full-scan acquisition

112

Analysis of Endocrine Disrupting Compounds in Food

mode and accurate mass analysis capabilities. Unambiguous identification is accomplished by means of accurate mass measurements from the (de)protonated molecules, in source collision-induced dissociation (CID) fragment ions, and isotope signature matching. Because LC-TOF-MS has the ability to record an unlimited number of compounds because it operates in full-scan mode, this technique is very convenient for the development of screening strategies based on the use of accurate mass databases. Recently, Mezcua et al. (2008) developed and evaluated a rapid automated screening method for the detection of pesticide residues (including organochlorine ED pesticides) in food using LC-TOF-MS, based on the use of an accurate mass database. The accurate mass database created by the authors includes data not only on the accurate masses of the target ions (300 pesticides) but also the characteristic in-source fragment ions (over 400 fragments included) and retention time data. The number of compounds that can be screened in an LC-TOFMS run can be easily upgraded (nontarget capabilities), thus enabling the reevaluation of the recorder data. Electrospray ionization (ESI) is the ionization source most frequently used for the LC-MS/MS analysis of pesticides in food. Chromatographic separation is, in general, achieved by means of C18 columns using acetonitrile or methanol and water as the mobile phase. To increase retention efficiency and MS sensitivity, mobile phase modifiers, buffers, and acids are recommended and widely used in concentrations from 2 to 20 mM. Formic acid is the modifier most widely used to analyze the studied EDCs in vegetables, fruits, and cereals (see Table 4.2). Because GC and LC are complementary techniques, some studies have combined both techniques to analyze a larger number of compounds. Payá et al. (2007) employed a combination of GC-QqQ-MS/MS and LCQLIT-MS/MS for the analysis of 80 pesti-

cides belonging to various chemical classes (including organochlorine ED pesticides) from various types of food commodities following extraction with the QuEChERS method. Diez et al. (2006) employed both GC-TOF-MS and LC-MS/MS for the analysis of a wide range of pesticides following the QuEChERS method. Ferrer et al. (2005) used GC-MS and LC-MS/MS for the identification and quantitation of pesticides olives and olive oil samples. A critical aspect in quantitative analysis with LC-MS and GC-MS is the occurrence of matrix effects. Each compound may be affected to a different extent by this phenomenon in complex matrices such as food, even from sample to sample; therefore, quantitation of the compounds is made by internal standard method, by matrix-matched standard calibration, or to a lesser extent, by standard addition method (see Table 4.2). Payá et al. (2007) used analyte protectants during GC analysis, and the use of these analytes have demonstrated a good alternative to the use of matrix-matched standards to minimize matrix effect–related errors.

Analysis of chlorinated endocrinedisrupting pesticides in products of animal origin Halogenated pollutants are lipophilic and stored in the body lipids in biota. In lipid-rich biota, the majority of pollutants may be stored in the depot lipids, whereas in lean biota (<1% lipids) the pollutants are also stored in the phospholipids. Because organochlorine pesticides do not readily degrade in the environment and are lipophilic, with a tendency to bioaccumulate, they can be found at high concentrations in fatty food, especially meats and fish. For products of animal origin, target compounds are, in most cases, those pesticides that accumulate in fat. Therefore, the polarity range is more limited than in fruits and vegetables.

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

Determination of chlorinated ED pesticides in products of animal origin is often complicated by the presence of different fat content. Methods for the isolation of lipid fractions from food products depend on the type of sample. The first step involves isolation of fat from the matrix by either extraction (milk, egg) or by rendering the fat (meat and fish). The fat can then be dissolved in a suitable solvent and separated from the analytes. Butter, fats, and oils are generally assumed to be homogeneous and normally do not require extensive extraction procedures. Aliquots of such samples can be dissolved in n-hexane or petroleum ether to the desired concentration. Meat and fish products having a lipid content of approximately 10 wt.% or lower are initially blended and homogenized. Next, a representative test sample is ground with anhydrous sodium sulfate until a free-flowing powder is obtained or lyophilized (Pang et al. 2006). Milk can either be freeze-dried or chemically dried with anhydrous sodium sulfate or subjected to a LLE procedure consisting of mixing with sodium oxalate and ethanol or methanol, followed by (repeated) extraction steps with a combination of organic solvents (Tsumura et al. 2002). Other authors initially separate fat by centrifugation (Ye et al. 2008). In the analysis of eggs, normally the egg yolk and white are separated for fat extraction from the egg yolk (Traag et al. 2006) Table 4.3 gives an overview of the analytical techniques currently employed to determine organochlorine endocrine-disrupter pesticides in products of animal origin.

Sample preparation Usually, the conventional approach to analyzing chlorinated pesticides in products of animal origin involves extraction of target analytes followed by cleanup of the obtained extract and gas chromatographic analysis.

113

The more widely used extraction technique to extract organochlorine pesticides in products of animal origin is solvent extraction (Janouskova et al. 2005; Pang et al. 2006; Waliszewski et al. 1997; Lehotay et al. 2001; Windal et al. 2009). Extraction of lipid-rich materials (mainly triglycerides) may be performed using a nonpolar solvent only (e.g., n-hexane, n-pentane) (Windal et al. 2009), but lean biological tissues require the use of medium polar (binary) solvent mixtures such as petroleum ether-acetone (Janouskova et al. 2005), acetone-acetonitrile (Pagliuca et al. 2005), or cyclohexane-ethyl acetate (Pang et al. 2006) to extract the halogenated pesticides from the phospholipids. Soxhlet extraction and sonication (Chen et al. 2007) have also been widely used. However, most of these methods are time consuming and use large quantities of organic solvents to remove the fatty materials. More recently, these traditional extraction methods are being replaced by rapid extraction methods such as SFE, MAE, MSPD, ASE, and SPME. SFE is described to provide cleaner extracts, less solvent handling, and equivalent or better recoveries than conventional solvent extraction techniques. Supercritical CO2 has been the most commonly used fluid for SFE because of its low critical constants, its low toxicity and cost, and its ability to extract quantitatively a wide range of relatively nonpolar organics from a variety of matrices. This method provides for the minimization of extraction of lipids and leads to direct GC analysis. However, in fatty food the pesticide residue still needs to be separated from the coextracted lipid matter. Some authors clean up the pesticide residues from extracted fat by using an alumina or silica column after SFE (Hopper 1999). Despite the fact that SFE of organochlorine ED pesticides from aqueous samples has shown remarkable advantages over solvent extraction techniques, there are indications that this technique is not yet completely successful, especially for biotic matrices. Hopper (1999) has developed an

Table 4.3. Overview of representative analytical techniques used for the determination of organochlorine endocrine-disrupter pesticides in products of animal origin.

Commodity Categories

Products of Animal Origin Sample Matrix

Seafood

Hexaclorobenzene, endrin, aldrin, chlordane, heptachlor … HCH, DDT, heptachlor, heptachlor epoxide, aldrin, dieldrin, and endrin

GPC mobile phase addition (ethyl acetate: cyclohexane) Fat removed from the abdomen of the fish

GPC mobile phase addition (ethyl acetate-cyclohexane)

Meat (beef, pork, rabbit, minced chicken) Meat (beef, mutton, pork, chicken, and rabbit)

Hexachlorobenzene, endrin, aldrin, chlordane, heptachlor… Chlordane: alphaChlordane: betachlordane and oxychlordane Heptachlor epoxide, simazine, atrazine, aldrin, lindane, linuron, dieldrin, alachlor, chlordane

Butter Butter and milk

Fish fat

Milk

Milk, cheese, yogurt, cream, butter

Meat

Eggs

Eggs

Eggs Eggs

Cleanup/ Derivatization GPC Ethyl acetate: cyclohexane

Ultrasonic water bath Petroleum ether

SPE (Florisil) Ethyl acetate and petroleum ether GPC Ethyl acetate: cyclohexane

Comminuted and homogenized

Solvent extraction Petroleum ether: acetone

GPC Florisil

Homogenized, blended, mixed with Na2SO4

Solvent extraction Cyclohexane: ethyl acetate

GPC Bio-beads S-X3 Cyclohexane-ethyl acetate

Cypermethrin, atrazine

Melted

SFE

SPE (Florisil)

HCB, aldrin, heptachlor, heptachlor epoxide, DDT, and endosulfan Lindane, acetochlor, alachlor,aldrin, heptachlor, DDT, endrin, dieldrin, chlordane… Chlordane: alphaChlordane, betachlordane, and oxychlordane

Milk fat layer separated by centrifugation Diluted in Milli-Q water

Solvent extraction Petroleum ether

Chlordane: alphaChlordane, betachlordane, and oxychlordane BHC, heptachlor, aldrin, chlordane, DDT, endosulfan, endrin,… DDT, atrazine, endosulfan, lindane… Lindane, HCB, heptachlor, aldrin, dieldrin, chlordane,endrin, nitrofen, DDT

Comminuted and homogenized

SPME Polydimethylsiloxane/ divinylbenzene fiber LLE Dichloromethane

GPC Florisil

Comminuted and homogenized

LLE Dichloromethane

GPC Florisil

Blended after removing and discarding shells

MSPD (Florisil) Dichloromethane: hexane Solvent extraction Acetonitrile Solvent extraction Hexane

Concentrated acid sulfuric

Fat extracted: hexane and acetone

i, Internal calibration; e, external calibration; s, with surrogate standard.

114

Extraction Technique (Sorbent) Elution Solvents

Pork fat

Milk and Milk Products

Preparation Method Pretreatment

Fish oil

Eggs

Compounds

SPE (Alumina) Hexane

Products of Animal Origin Analytical Method Detection Mode

LC or GC Column Mobile Phase

LODs (μg/Kg or μg/L*)

LOQs (μg/Kg or μg/L*)

Recovery (%)

Reference

GC-QqQ-MS/MS (EI), MRM

VF-5MS

0.1–2*

65–103 i,e

Patel et al. 2005

Two-dimensional GC/ECD

DB-1701 HP-5MS

0.6–8

81.4–90.4 e

Chen et al. 2007

GC-QqQ-MS/MS (EI), MRM

VF-5MS

0.1–2

64–101 i,e

Patel et al. 2005

GC-ECD Two ECD

DB-5 DB-17

0.002–0.05

i

Janouskova et al. 2005

GC-Q-MS SIM LC-QqQ-MS/MS API,(ESI+), MRM

GC-Q-MS: DB-1701 LC-QqQ-MS/MS:C18 Water/Acetonitrile

0.4–600

40–120 i, e

Pang et al. 2006

GC-ELCD (organochlorine) GC-ECD

DB-1 8 (organochlorine) Fused silica capillary column

0–118

Hopper et al.1999

71.3–132.8 e

Waliszewski et al. 1997

GC-ECD

HP-5

0.04–0.9*

69–122 i, e

Fernández-Alvarez et al. 2008

GC-ECD Two ECD

DB-5 DB-17

0.002–0.05

i

Janouskova et al. 2005

GC-ECD Two ECD

DB-5 DB-17

0.002–0.05

i

Janouskova et al. 2005

GC-ECD GC-Q-MS (EI) SIM

DB-5 DB-5-MS

0.2–0.7

0.7–2.3

82–110 i

Valsamaki et al. 2006

(DSI)GC-IT-MS/MS

Rtx-5MS

0.01–0.1

53–123 e

Lehotay et al. 2001

GC-ECD Two GC columns and two ECD

HT-8 DB1701

i, s

Windal et al. 2009

0.2–600

1–3 0.003–0.56*

10*

115

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Analysis of Endocrine Disrupting Compounds in Food

automated SFE and in-line cleanup system for organochlorine and organophosphate pesticide residues contained in fatty food, including butterfat. This procedure uses supercritical carbon dioxide modified with 3% acetonitrile at 27–58 MPa and 60°C to extract and separate the pesticide residues from the fat on a C1bonded phase preparative column at 95°C. The automated C1 system recovers 86 of 117 nonpolar to moderately polar organochlorine and organophosphate pesticides from fats. Matrix solid-phase dispersion is a relatively recent extraction and cleanup technique used for the simultaneous determination of various pollutants from semisolid and solid samples and has been almost exclusively applied to the analysis in food commodities. Although some MSPD extracts are clean enough to be subjected to instrumental analysis directly, a cleanup step is often required. Additionally, with fatty substrates rigorous cleanup is necessary for satisfactory peak separation, sensitivity, and overall performance of the chromatographic system. Thus, a cleanup of the extracts is usually needed, especially to separate the low-polarity analytes such as organochlorine ED pesticides. Cleanup methods for fatty samples quoted in the literature include shaking with concentrated sulfuric acid, silica gel chromatography, acidified silica gel chromatography, Florisil chromatography, and alumina chromatography (Valsamaki et al. 2006). Valsamaki and coworkers have developed a multiresidue method for the determination of 20 organochlorine pesticides and eight PCB congeners in chicken eggs using MSPD with Florisil as the sorbent material and dichloromethane-hexane as the eluting solvent. Further purification of the extracts was conducted using a conventional cleanup procedure with concentrated sulfuric acid followed by GC-ECD for the determination and quantification of the target analytes and GC-MS in SIM mode for confirmation. The detection limits were <0.7 ng/g for all com-

pounds, and the recoveries ranged from 82% to 110%. The performance of the concentrated sulfuric acid treatment compared to the alumina cleanup procedure was assessed by the authors with respect to its ability to remove lipidic material and to recover the target analytes. Alumina was discarded because of the low effectiveness of this cleanup, obtaining more dirty extracts than the conventional cleanup method using sulfuric acid. Recently, solid-phase microextraction methods have been developed for the rapid determination of chlorinated pesticides in aqueous samples such as milk (FeranándezAlvarez et al. 2008). In these studies, chlorinated pesticides can be effectively extracted without solvent, and cleanup procedure can be eliminated for the removal of interferences. Fernández-Alvarez and coworkers have developed a simple and rapid method based on SPME technique followed by gas chromatography with microelectron-capture detection for the simultaneous determination of more than 30 pesticides (pyrethroids and organochlorine among others) in milk. Negative matrix effects due to the complexity and lipophilicity of the matrix were reduced by diluting the sample with distilled water. The optimization of the extraction stage was carried out by fractional factorial design. Results showed that the sampling mode, stirring, and temperature were the most significant variables affecting extraction efficiency. The use of the multivariate strategy for optimization demonstrated that several factor interactions were also significant for most of the target pesticides. The optimal experimental conditions implied the use of polydimethylsiloxane/divinylbenzene coating for direct extraction at 100°C of 1 mL of milk sample diluted 1:10 with Milli-Q water and stirring for 30 min. LODs and limits of quantitation (LOQs) were satisfactory and complied with the current maximum residue limits of the target pesticides in milk and dairy products.

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

LLE is the most frequently used methodology for the extraction of halogenated pesticides in liquid samples such as eggs and milk (Janouskova et al. 2005). The main difficulty with the extraction of products of animal origin is the coextraction of lipid components from matrix that is incompatible with the GC systems. A critical step, and generally the most laborious aspect in the analytical procedure, is the cleanup of the extracts. Therefore, a variety of different cleanup methods have been studied in the literature, such as LLE, GPC, low-temperature precipitation, and adsorption chromatography on different sorbents (silica, alumina, Florisil). Florisil is the most popular sorbent employed today because it is particularly suited for fatty foods (Chen et al. 2007; Windal et al. 2009). GPC is probably the most widely applied technique (Janouskova et al. 2005; Pang et al. 2006; Patel et al. 2005). Important advantages of this technique are that it can be easily automated and has a large scope for pesticide residue analysis, because separation occurs on the basis of molecular size. Hence, relatively large molecules such as chlorophylls and triglycerides are separated from the lower-molecularmass pesticides. An important drawback of GPC is that the triglycerides, which are usually present at the milligram level, elute before the pesticides, which are present at the microgram to nanogram level. Some tailing of the large triglyceride peak is inevitable and makes on-line combinations of GPC and capillary GC difficult to realize. To overcome this problem, some authors suggest an additional SPE cleanup step. Although this provides cleaner extracts, it can also result in low recoveries for some organochlorine pesticides, and in addition, the increase in time and solvent usage makes this option less desirable. This need can be overcome by using a selective detector, such as tandem mass spectrometry (MS/MS).

117

Instrumental analysis and quantitation GC is the method of choice for the analysis of organochlorine ED pesticides in the complex animal origin matrices. Most GC methods employ the element-selective detector ECD for the detection of the studied compounds in these types of samples because of its high resolution and good sensitivity in the nanogram range (Waliszewski et al. 1997; Fernández-Alvarez et al. 2008; Janouskova et al. 2005; Valsamaki et al. 2006; Windal et al. 2009). Other advantages of this detector include reduced cost of operation and the fact that it requires less technical skill to obtain reliable results. The ECD detectors are sometimes connected in parallel to allow results to be obtained with only one injection (Janouskova et al. 2005). Nevertheless, analytical problems associated with the analysis of pesticides in fatty matrices are well known and the detectors mentioned above do not provide unequivocal confirmation of identity and are often subject to matrix interferences. Mass spectrometry (MS), usually in the selected ion monitoring (SIM) mode, is also widely employed for the determination of organochlorine ED pesticides in complex matrices (Pang et al. 2006). It has better resolution and can give higher sensitivity than the ordinary GC method, although confidence in the confirmation of identity may be reduced if one or more of the selected ions are affected by matrix interferences, giving poor spectral information. Alternatively, MS/MS with ion trap or triple quadrupole can be employed to achieve a high level of selectivity and low detection limits in dirty extracts (Lehotay et al. 2001; Windal et al. 2009; Patel et al. 2005). Comprehensive GCxGC has proven to be a strong technique for separating complex mixtures and provides considerably more information on the pollutant profile when compared to traditional GC (Chen et al. 2007). In addition, GCxGC is also excellent for the identification of unknown compounds

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Analysis of Endocrine Disrupting Compounds in Food

appearing (or interfering) in the chromatogram. In GCxGC analysis, TOF-MS and ECD were used for the detection of organochlorine pesticides (Chen et al. 2007). With GC–ECD and/or GC–MS determinations, organochlorine pesticides are typically separated on nonpolar (e.g., DB-5, DB-5MS HP-5, RTx-5MS, HP-5MS, DB-1, VF-5MS) or slightly polar stationary phases with dimensions of 30–60 m × 0.25 mm I.D., 0.25 m film thickness. Applying two columns in parallel, using a single injection with splitting to the two columns ending in two ECD detectors was used to separate organochlorine pesticides and PCB isomers in eggs (Windal et al. 2009). The splitless injection technique for introducing the sample into the GC is generally preferred over on-column injection due to its cleanliness. Recently, the use of the programmable temperature vaporizer (PTV) injector is gaining acceptance. The more interesting application of PTV is that of large-volume injection (LVI) for increasing the sensitivity. Using this technique, volumes of 10–50 μL may be injected (van Leeuwen and de Boer 2008). Lehotay and coworkers (2001) have employed direct sample introduction (DSI)/ GC-MS-MS to extract pesticide residues, including organochlorine pesticides, in eggs. DSI, or “dirty sample injection,” is a rapid, rugged, and inexpensive approach to largevolume injection in GC. This technique greatly minimizes sample preparation and yet still provides a rugged analytical approach for complex matrices. A major benefit in this DSI approach is that nonvolatile matrix components, which normally contaminate the GC liner and column in traditional injection approaches, remain in the microvial, which is disposed of after every injection. DSI of complex samples such as eggs requires a very selective detection technique, such as tandem mass spectrometry (MS−MS), to determine the analytes among the many semivolatile matrix components that also appear. In this

study, experiments involved no cleanup or cleanup using C18 or PSA SPE cartridges, addition or no addition of MgSO4, use of internal standards or no internal standards, and minor alteration of instrument conditions. The results for the majority of the targeted pesticides were acceptable in all experiments, but certain pesticides gave irreproducible results independent of the changes made to the method. Ultimately, the final method was streamlined to provide the fastest and easiest overall procedure by eliminating SPE cleanup, extended solvent evaporation time, addition of MgSO4, and use of internal standards. Good results were obtained for approximately 25 pesticides, and inadequate sensitivity was achieved for the others. In the case of the organochlorine pesticide studied (p,p′-DDE, p,p′-DDT, atrazine, endosulfan alpha and beta, and lindane), the recoveries were higher than 60%; only the endosulfan sulfate was not recovered. Accurate analysis of halogenated pollutants is important for scientists and policy makers who rely on the data produced in environmental laboratories. To minimize the chance of errors, steps should be taken to improve the analysis and quality control systems should be established and routinely applied, including the use of high-quality standards and internal standards, blank tests, replicate analysis, recovery experiments, and plotting of quality control charts. The selectivity of ECD detectors is limited: 13 C12-labeled standards cannot be used and coelutions and other interferences can cause biased results. MS techniques are preferred for accurate determination because of the unambiguous identification and because 13 C12-labeled standards can be used. Besides isotopic-labeled standards, to increase accuracy of results (FernándezAlvarez et al. 2008; Valsamaki et al. 2006) matrix-matched calibration has also been employed in the analysis of organochlorine ED pesticides in products of animal origin (Chen et al. 2007; Waliszewski et al. 1997; Lehotay et al. 2001).

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

Occurrence of organochlorine endocrine-disrupter pesticides in food commodities

ticides accumulate in different environmental compartments and in food chains. These residues bioconcentrate in lipid-rich tissues according to the equilibrium pattern of internal transport and lipid tissue content and decline at a very slow rate, even after sources of contamination are eliminated. On the other hand, most organochlorine herbicides and fungicides, which are usually more polar compounds than previously commented, are commonly used today. Figure 4.2 shows the occurrence and the mean concentration of the most frequent organochlorine endocrine-disrupter pesticides in fruits and vegetables with high water and high oil content and products of animal origin.

Organochlorine pesticides have been used in agriculture and livestock for a long time to control a variety of crop diseases and pests. They have also been employed to combat vectors of malaria and some other deadly diseases of humans. Of late, some of these compounds, which are also constituents of the toxic group known as persistent organic pollutants (POPs), have been banned or restricted. However, owing to their highly persistent nature and due to their chemical stability and lipophilic character, the organochlorine pes-

HIGH OIL CONTENT FRUITS AND VEGETABLES

HIGH WATER CONTENT FRUITS AND VEGETABLES DDT,DDE and metabolites 0.2 mg// kg

Others 22 %

Others

Diuron 0.13 mg/kg /

4% 4%

HCB

11 % 14 %

Iprodione

119

0.4 mg/kg

11 %

2.5 mg/kg

Endosulfan α, β and sulfate

42 %

94 %

0.015 mg/kg

Endosulfan α, β and sulfate 1.3 mg/kg

PRODUCTS OF ANIMAL ORIGIN Chlordane isomers 0.019 mg/kg HCHs 7%

0.052 mg/kg

34 % 23 %

13 %

DDT and DEE 1.16 mg/kg

23 %

Endosulfan α, β and sulfate 0.007 mg/kg

HCB 0.091 mg/kg

Figure 4.2. Occurrence of organochlorine endocrine-disrupter pesticides in fruits and vegetables with high water and high oil content and products of animal origin. Data of average concentration of pesticides residues in milligrams per kilogram are also included.

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Analysis of Endocrine Disrupting Compounds in Food

Fruits and vegetables Fruits and vegetables with high water content Endosulfan is the most commonly detected organochlorine ED pesticide in fruits and vegetables, presumably due to ongoing use of this pesticide. Although it is being phased out in most countries of the European Union, endosulfan continues to have wide use as an insecticide across the globe. In fruits and vegetables with high water content, endosulfan has been found in tomato juice, pepper, onion, celery, cabbage, and other fruits and vegetables, with a mean concentration of 1.3 mg/kg. Hexachlorobenzene (HCB) residues were detected in cabbage, celery, and other vegetables and fruits at mean levels of 0.4 mg/kg. HCB is known as a by-product of some industrial chlorination and combustion processes; therefore, its presence in food products could be due to industrial contamination. Because of restrictions and bans of the chemical, levels of HCB have declined. Iprodione is a fungicide widely used in fruits and other crops. In the revised literature it was found in pear, apple, grape, and some vegetables at mean concentrations of 2.5 mg/kg. The persistent DDT and its main and extremely stable metabolite (DEE), were detected in some fruits and vegetables. Although many developed nations restricted or banned DDT in the 1970s, restrictions in the developing world were not common until the 1980s, and in some parts of the world DDT continues to be used in disease vector control (e.g., malaria). This use is due to its low cost and the unavailability of viable alternatives, hence in these countries DDT and DEE concentrations in the environment and animals remain high. Other target compounds found occasionally in fruits and vegetables are chlordane, prochloraz, dieldrin, heptachlor, acetachlor, and lindane.

Fruits and vegetables with high oil content In the case of high oil-content samples, the matrices studied are mainly olives and olive oil, and endosulfan was about the only target compound detected, at a mean level of 0.015 mg/kg. In addition to endosulfan, although less frequently, some herbicides have been found in olives and olive oil, diuron being the most important, with a mean concentration of 0.13 mg/kg. It was observed that, in general, herbicides were found with a higher concentration in oil coming from soil olives than in samples coming from flight olives (Guardia-Rubio 2006). This result is reasonable considering that herbicides are applied to the soil under the olive tree and can persist to the harvest stage, making possible the contamination of the olives when they fall down. In the case of endosulfan, the levels found for both types of olive oil samples were similar, and this result is consistent with the fact that endosulfan is applied directly to the olive tree.

Products of animal origin In products of animal origin, the presence of organochlorine ED pesticides are mainly found in fish, milk, butter, and eggs. Hexachlorobenzene has been detected in milk, butter, eggs, and fish at mean levels of 0.091 mg/kg. As noted previously, HCB is a by-product of some industrial chlorination processes, which is probably the main source of environmental and animal contamination. Hexachlorocyclohexane isomers (HCHs) were detected in milk, butter, and fish samples at a mean concentration of 0.052 mg/kg. Although the production of technical HCH is being phased out in most countries, it was reported that residues of HCH persist in the environment and accumulate in animal tissues and fluid due to their lipophilic nature. Among the main isomers of HCH (alpha, beta, and gamma), the most persistent and metaboli-

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

cally stable is the beta-isomer. However, the gamma-HCH (lindane) is the most active and important isomer. In most studies, the frequency of different isomers of HCH residues in dairy products on the basis of fat was of the order beta > alpha > gamma. In addition, with regard to mean concentration in the contaminated samples, beta-isomer was highest, followed by alpha-isomer and gamma-isomer (Waliszewski et al. 1997). The predominance of occurrence of beta-HCH is due to the isomerization of alpha- and gamma-HCH into beta-HCH in the environment and also due to its high stability. The ratios and levels of HCH isomers are often used as evidence of past or current HCH application. If the betaHCH is the most abundant isomer, this suggests rather older residues from use in the past rather than current usage of technical mixtures. Chlordane isomers were also detected in products of animal origin. The highest concentration of chlordane was found in freshwater fish, meat, and fats. Chlordane is a broad-spectrum contact insecticide that has been applied to agricultural crops, and it has also been used extensively to eradicate termites. This compound is almost insoluble in water and soluble in organic solvents; it is highly stable and semivolatile. Analogous to other chlorinated pesticides, these substances are also deposited in the fat tissues of animals and are very slowly metabolized and excreted from the body. DDT and DDE, its main metabolite, are the organochlorine endocrine-disrupter pesticides most frequently found and at the highest mean concentration (1.16 mg/kg) in fish, meat, eggs, butter, cheese, and milk. Windal et al. (2009) detected DDT or DDE in 95% of the home-produced eggs analyzed, and 17% of the samples were above the norm (in Belgium) for DDT (500 ng/g fat). These high levels raise questions about the presence of DDT banned since 1976. No direct correlation could be established between soil and egg concentration for DDT. However, the

121

contact with soil and the environment is probably responsible for the higher concentrations found in home-produced eggs compared to commercial production of eggs for which usually no organochlorine pesticides can be detected. In some cases, the historical uses of DDT for henhouse treatment against ectoparasites can explain the high levels found in eggs. DDT is rapidly metabolized to DDE in a hen’s body and is excreted via the egg. In agreement with this observation, 60–100% of the total DDT in an egg is due to the p,p′-DEE isomer, and the rest is due to the p,p′-DDT isomer (Windal et al. 2009). In most of the recent studies in developed nations, p,p′DDE was the only metabolite among the DDTs that was detected in eggs. However, DDT was not often detected. This is due to the fact that there is no actual use of DDT in recent times. When DDT was sprayed, it drifted for long distances and evaporated or attached to windblown dust. In the environment and animals, DDT breaks down to p,p′DEE, an extremely stable compound that resists further environmental breakdown or metabolism by organisms. DDT and DDE are highly soluble in lipids, and concentrations of these compounds showed age dependency. It may take between 10 and 20 years for DDT to disappear from an exposed individual, but DDE would possibly persist throughout that individual’s life span. Low concentrations of endosulfan (0.007 mg/kg) were found in milk and butter. Endosulfan is a very popular and cheap insecticide used for management of a variety of insect pests in a wide range of crops. Although the transfer coefficient of endosulfan from feed to milk and other products of animal origin is very low, its residues could be found in these samples. The presence and concentration of HCHs, DDT, chlordane isomers, and HCB residues in products of animal origin indicate that although the frequency and level has considerably decreased, the contamination still exists, albeit in low scale. The most important

122

Analysis of Endocrine Disrupting Compounds in Food

reason for this could be that most of the organochlorine compounds have become persistent environmental contaminants over the decades and have less degradability. They therefore would require much more time to be completely phased out from the system. On the other hand, some of the compounds such as DDT or lindane are not totally banned, but rather are restricted in some countries, and animals may still be exposed to these compounds. Although the target compound levels found in the reviewed literature seems to be too low to pose acute or chronic toxic effects on the basis of current toxicological knowledge, we have to consider the potential of these chemicals to be endocrine disrupters at low concentration levels, and synergistic effects among them cannot be excluded.

Acknowledgments The authors thank the Spanish Ministry of Education and Science (contract CTM200765544/TECNO) and the Junta de Andalucía (Project ref. AGR-4047) for their financial assistance. María José Gómez acknowledges the Juan de la Cierva research contract from the Spanish Ministry of Science and Technology.

References Abhilash P.C., Singh V., Singh N. Simplified determination of combined residues of lindane and other HCH isomers in vegetables, fruits, wheat, pulses, and medicinal plants by matrix solid-phase dispersion (MSPD) followed by GC-ECD. Food Chem., 113 (2009) 267–371. Ahmed F.E. Analyses of pesticides and their metabolites in foods and drinks. Trends Anal. Chem., 20, no. 11 (2001) 649–661. Albero B., Sánchez-Brunete C., Donoso A., Tadeo J.L. Determination of herbicide residues in juice by solidphase dispersion and gas chromatography-mass spectrometry. J. Chromatogr. A., 1043 (2004) 127–133. Albero B., Sánchez-Brunete C., Tadeo J.L. Determination of endosulfan isomers and endosulfan sulfate in tomato juice by matrix solid-phase dispersion and gas chromatography. J. Chromatogr. A., 1007 (2003) 137–143.

Albero B., Sánchez-Brunete C., Tadeo J.L. Multiresidue determination of pesticides in juice by solid-phase extraction and gas chromatography-mass spectrometry. Talanta, 66 (2005) 917–924. Amvrazi E.G., Albanis T.A. Pesticide residue assessment in different types of olive oil and preliminary exposure assessment of Greek consumers to the pesticide residues detected. Food Chem., 113 (2009) 253–261. Anastassiades M., Lehotay S.J., Stajnbaher D., Schenck F.J. QuEChERS: A new sample preparation technique for multiresidue analysis of pesticides in foods and agricultural samples. J. AOAC Int., 86 (2003) 412–431. Ballesteros-Gómez A., Rubio S., Pérez-Bendito D. Analytical methods for the determination of bisphenol A in food. J. Chromatogr. A. 1216 (2009) 449–469. Barnerjee K., Patil S.H., Dasguptap S., Oulkar D.P., Patil S.B., Savant R., Adsule P.G. Optimization of separation and detection conditions for the multiresidue analysis of pesticides in grapes by comprehensive twodimensional gas chromatography-time-of-flight mass spectrometry. J. Chromatogr. A., 1190 (2008) 350–357. Beyer A., Biziuk M. Applications of sample preparation techniques in the analysis of pesticides and PCBs in food. Food Chem., 108 (2008) 669–680. Björklund E., Müller A., von Holst C. Comparison of fat retainers in accelerated solvent extraction for the selective extraction of PCBs from fat-containing samples. Anal. Chem. 73 (2001) 4050–4053. BKH Consulting for European Commission DG Env. (EC-BKH). 2000. Towards the Establishment of a Priority List of Substances for Further Evaluation of Their Role in Endocrine Disruption. Available from ec.euroopa.eu/environment/docum/pdf/bkh_main. pdf. Blasco C., Picó Y. Liquid chromatography-mass spectrometry. Food Toxicants Analysis, Chapter 14. New York, Elsevier Science (2007) 509–559. Boenke A. Contribution of European research to antimicrobials and hormones. Anal. Chim. Acta. 473 (2002) 83–87. Boti V.I., Sakkas V.A., Albanis T.A. An experimental design approach employing artificial neural networks for the determination of potential endocrine disruptors in food using matrix solid-phase dispersion. J. Chromatogr. A., 1216 (2009) 1296–1304. Boti V.I., Sakkas V.A., Albanis T.A. Measurement uncertainty arising from trueness of the analysis of two endocrine disruptors and their metabolites in environmental samples. Part I: Ultrasonic extraction from diverse sediment matrices. J. Chromatogr. A., 1146 (2007) 139–147. Cajka T., Hajslova J., Lacina O., Mastovska K., Lehotay S.J. Rapid analysis of multiple pesticide residues in fruit-based baby food using programmed temperature vaporiser injection–low-pressure gas chromatography–high-resolution time-of-flight mass spectrometry. J. Chromatogr. A., 1186 (2008) 281– 294.

Analysis of Organochlorine Endocrine-Disrupter Pesticides in Food Commodities

Chen S., Shi L., Shan Z., Hu Q. Determination of organochlorine pesticide residues in rice and human and fish fat by simplified two-dimensional gas chromatography Food Chem., 104 (2007) 1315–1319. Colborn T, Dumanoski D, Meyers J.P. Our Stolen Future: Are We Threatening Our Fertility, Intelligence and Survival? New York, Dutton Adult. 1996 Columé A., Cárdenas S., Gallego M., Valcárcel M. Semiautomatic method for the screening and determination of 23 organochlorine pesticides in horticultural samples by gas chromatography with electroncapture detection. J. Chromatogr. A., 849 (1999) 235–243. Dallüge J., Rijn M., Beens J., Vreuls R., Brinkman U. Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometric detection applied to the determination of pesticides in food extracts. J. Chromatogr. A., 965 (2002) 207–217. David F., Sandra P. Stir bar sorptive extraction for trace analysis. J. Chromatogr. A., 1152 (2007) 54–69. Diagne R.G., Foster G.D., Khan S.U. Comparison of Soxhlet and microwave-assisted extractions for the determination of fenitrothion residues in beans. J. Agric. Food Chem., 50 (2002) 3204–3207. Diamanti-Kandarakis E., Bourguignon J.-P., Giudice L.C., Hauser R., Prins G.S., Soto A.M., Zoeller R.T., Gore A.C. Endocrine-disrupting chemicals: An Endocrine Society Scientific statement. Endocri. Rev. 30 (2009) 293–342. Díez C., Traag W.A., Zommer P., Marinero P., Atienza J. Comparison of an acetonitrile extraction/partitioning and “dispersive solid-phase extraction” method with classical multi-residue methods for the extraction of herbicide residues in barley samples. J. Chromatogr. A., 1131 (2006) 11–23. European Commission. 2006. SANCO/10232/2006. Available from ec.europa.eu/food/plant/resources/ qualcontrol_en.pdf. European Union (EU). Commission Regulation (EC) 208/2005. Off. J. Eur. Comm. L., 34 (2005) 3. Fernandez-Alvarez M., Llompart M., Lamas J.P., Lores M., Garcia-Jares C., Cela R., Dagnac T. Development of a solid-phase microextraction gas chromatography with microelectron-capture detection method for a multiresidue analysis of pesticides in bovine milk. Ana. Chim. Acta, 617 (2008) 37–50. Ferrer C., Gómez M.J., García-Reyes J.F., Ferrer I., Thurman M., Fernández-Alba A.R. Determination of pesticide residues in olives and olive oil by matrix solid-phase dispersion followed by gas chromatrography/tandem mass spectrometry. J. Chromatogr. A., 1069 (2005) 183–194. Fong W.G., Moye H.A., Seiber J.N., Toth J.P. Pesticide Residues in Foods. Methods, Techniques, and Regulations. New York: John Wiley & Sons. (1999) 339. Fussell R.J., Addie K.J., Reynolds S.L., Wilson M.F. Assessment of the stability of pesticides during cryogenic sample processing. 1. Apples. J. Agric. Food Chem., 50 (2002) 441–448. García-Reyes J.F., Ferrer C., Gómez-Ramos M.J., Molina-Díaz A., Fernández-Alba A.R. Determination

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of pesticide residues in olive oil and olives. Trends Anal. Chem., 26 No. 3 (2007) 239–251. Gilbert-López B., García-Reyes J.F., Molina-Díaz A. Sample treatment and determination of pesticide residues in fatty vegetable matrices: A review. Talanta, 79 (2009) 109–128. González-Rodríguez R.M., Rial-Otero R., CanchoGrande B., Simal-Gándara J. Determination of 23 pesticide residues in leafy vegetables using gas chromatography-ion trap mass spectrometry and analyte protectants. J. Chromatogr. A., 1196–1197 (2008) 100–109. Guardia-Rubio M., Fernandez-De Cordova M.L., Ayora-Cañada M.J., Ruiz-Medina A. Simplified pesticide multiresidue analysis in virgin olive oil by gas chromatography with thermoionic specific, electron-capture, and mass spectrometric detection. J. Chromatogr. A., 1108 (2006) 231–239. Hercegová A., Dömötörová M., Matisová E. Sample preparation methods in the analysis of pesticide residues in baby food with subsequent chromatographic determination. J. Chromatogr. A., 1153 (2007) 54–73. Hernández F., Pozo O.J., Sancho J.V., Bijlsma L., Barreda M., Pitarch E. Multiresidue liquid chromatography tandem mass spectrometry determination of 52 non-gas chromatography-amenable pesticides and metabolites in different food commodities. J. Chromatogr. A., 1109 (2006) 242–252. Hiemstra M., Kok A. Comprehensive multi-residue method for the target analysis of pesticides in crops using liquid chromatography-tandem mass spectrometry. J. Chromatogr. A., 1154 (2007) 3–25. Hopper M.L. Automated one-step supercritical fluid extraction and clean-up system for the analysis of pesticide residues and fatty matrices. J. Chromatogr. A., 840 (1999) 93–105. Huskova R., Matisova E., Svorc L., Mocak J., Kirchner M. Comparison of negative chemical ionization and electron impact ionization in gas chromatographymass spectrometry of endocrine disrupting pesticides. J. Chromatogr. A., 1216 (2009) 4927–4932. Institute for Environment and Health (IEH), Environmental oestrogens: Consequences to human health and wildlife. Leicester, UK: IEH. (1995). IEH 2005. http://www.cranfield.ac.uk/health/researchareas/ environmenthealth/ieh/ieh%20publications/w20.pdf. Janouskova E., Krbuskova M., Rehurkova I., Klimova M., Prokes L., Ruprich J. Determination of chlordane in foods by gas chromatography. Food Chem., 93 (2005) 161–169. Kmellár B., Fodor P., Pareja L., Ferrer C., Martínez-Uroz M.A., Valverde A., Fernández-Alba A.R. Validation and uncertainty study of a comprehensive list of 160 pesticide residues in multi-class vegetables by liquid chromatography-tandem mass spectrometry. J. Chromatogr. A., 1215 (2008) 37–50. Knezevic Z., Sedar M. Screening of fresh fruit and vegetables for pesticide residues on Croatian market. Food Control, 20 (2009) 419–422. Kristenson E.M., Haverkate E.G.J., Slooten C.J., Ramos L., Vreuls R.J.J., Brinkman U.A.T. Miniaturized

124

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automated matrix solid-phase dispersion extraction of pesticides in fruits followed by gas chromatographymass spectrometric analysis. J. Chromatogr. A., 917 (2001) 277–286. Lacorte S., Fernandez-Alba A.R. Time of flight mass spectrometry applied to the liquid chromatographic analysis of pesticides in water and food. 2006. Wiley InterScience DOI 10.1002/mas.20094. 2006. www.interscience.wiley.com. Lal A., Tan G., Chai M. Multiresidue analysis of pesticides in fruits and vegetables using solid-phase extraction and gas chromatography methods. Analytical Sciences, 24 (2008) 231–236. Lehotay S.J., Kok A., Hiemstra M., Bodegraven P.V. Validation of a fast and easy method for the determination of residues from 229 pesticides in fruits and vegetables using gas and liquid chromatography and mass spectrometric detection. J. AOAC Int., 88 (2005) 595–614. Lehotay S.J., Lightfield A.R., Harman-Fetcho J.A., Donoghue D.J. Analysis of pesticide residues in eggs by direct sample introduction/gas chromatography/ tandem mass spectrometry. J. Agric. Food Chem., 49 (2001) 4589–4596. Mansour S.A., Belal M.H., Abou-Arab A.A.K., Gad M.F. Monitoring of pesticides and heavy metals in cucumber fruits produced from different farming systems. Chemosphere, 75 (2009) 601– 609. Mezcua M., Ferrer C., García-Reyes J. F., MartínezBueno M.J., Albarracín M., Claret M., FernándezAlba A.R. Determination of selected non-authorized insecticides in peppers by liquid chromatography time-of-flight mass spectrometry and tandem mass spectrometry. Rapid Commun. Mass Spectrom., 22 (2008) 1384–1392. Mezcua M., Malato O., García-Reyes J.F., Molina-Díaz A., Fernández-Alba A.R. Accurate-mass databases for comprehensive screening pesticide residues in food by fast liquid chromatography time-of-flight mass spectrometry. Anal. Chem., 81 (2009) 913–929. Mezcua M., Martínez-Uroz M.A., Wylie P.L., FernándezAlba A.R. Simultaneous screening and target analytical approach by GC/quadrupole/MS for pesticide residues in fruits and vegetables. J. AOAC Int., 92 (2009) 1790. Nguyen T.D., Han E.M., Seo M.S., Kim S.R., Yun M.Y., Lee D.M., Lee G-H. A multi-residue method for the determination of 203 pesticides in rice paddies using gas chromatography/mass spectrometry. Ana. Chim. Acta, 619 (2008) 67–74. Okihashi M., Takatori S., Kitagawa Y., Tanaka Y. Simultaneous analysis of 260 pesticide residues in agricultural products by gas chromatography/triple quadrupole mass spectrometry. J. AOAC Int., 90 (2007) 1165–1179. Pagliuca G., Gazzotti T., Zironi E, and Sticca P. Residue analysis of organophosphorus pesticides in animal matrices by dual column capillary gas chromatography with nitrogen-phosphorus detection. J. Chromatogr. A, 1071 (2005) 67–70. Pang G.F., Cao Y.Z., Zhang J.J., Fan C.L., Liu Y.M., Li X.M., Jia G.Q., Li Z.Y., Shi Y.Q., Wu Y.P., Guo T.T.

Validation study on 660 pesticide residues in animal tissues by gel permeation chromatography cleanup/ gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. J. Chromatogr. A., 1125 (2006) 1–30. Patel K., Fussell R.J., Hetmanski M., Goodball D.M., Kelly B.J. Evaluation of gas chromatography-tandem quadrupole mass spectrometry for the determination of organochlorine pesticides in fats and oils. J. Chromatogr. A., 1068 (2005) 289–296. Payá P., Anastassiades M., Mack D., Sigalova I., Tasdelen B., Oliva J., Barba A. Analysis of pesticide residues using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) pesticide multiresidue method in combination with gas and liquid chromatography and tandem mass spectrometric detection. Anal Bioanal Chem., 389 (2007) 1697–1714. Pengyan L., Qingxue L., Yusong M., Jinwei L., Xuan J. Analysis of pesticide multiresidues in rice by gas chromatography-mass spectrometry coupled with solid phase extraction. Chin. J. Chromatogr., 24 (2006) 228–234. Pihlstrom T., Blomkvist G., Friman P., Pagard U., Osterdahl B.G. Analysis of pesticide residues in fruit and vegetables with ethyl acetate extraction using gas and liquid chromatography with tandem mass spectrometric detection. Anal Bioanal Chem., 389 (2007) 1773–1789. Ramos J.J., Rial-Otero R., Ramos L., Capelo J.L. Ultrasonic-assisted matrix solid-phase dispersion as an improved methodology for the determination of pesticides in fruits. J. Chromatogr. A., 1212 (2008) 145–149. Ridgway K., Lalljie S.P.D., Smith R.M.. Advances in Sample Preparation. Part II. Sample preparation techniques for the determination of trace residues and contaminants in foods. J. Chromatogr. A. 1153 (2007) 36–53. Schachterle S., Feigel C. Pesticide residue analysis in fresh produce by gas chromatography-tandem mass spectrometry. J. Chromatogr. A., 754 (1996) 411– 422. Tao C.J., Hu J.Y., Li J.Z., Zheng S.S., Liu W., Li C.J. Multi-residue determination of pesticides in vegetables by gas chromatography/ion trap mass spectrometry. Bull. Environ. Contam. Toxicol., 82 (2009) 111–115. Traag W.A., Kan C.A., van der Weg G., Onstenk C., Hoogenboom L.A.P. Residues of dioxins (PCDD/Fs) and PCBs in eggs, fat and livers of laying hens following consumption of contaminated feed. Chemosphere, 65 (2006) 1518–1525. Tsumura Y., Ogino T., Hirota N., Sone Y. Nutritional evaluation of take-out lunches available at convenience stores. Journal of Human Life Sciences. 1 (2002) 17–24. Twombly R.. Assault on the male. Environ Health Perspect. 103 (1995) 802–805. Valsamaki V.I., Boti V.I., Sakkas V.A., Albanis T.A. Determination of organochlorine pesticides and polychlorinated biphenyls in chicken eggs by matrix solid phase dispersion. Ana. Chim. Acta, 573–574 (2006) 195–201.

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van Leeuwen S.P.J., de Boer J. Advances in the gas chromatographic determination of persistent organic pollutants in the aquatic environment. J. Chromatogr. A. 1186 (2008) 161–162. Waliszewski S.M., Pardío V.T., Waliszewski K.N., Chantiri J.N., Aguirre A.A., Infanzón R.M., Rivera J. Organochlorine pesticide residues in cow’s milk and butter in Mexico. The Science of the Total Environment, 208 (1997) 127–132. Walorczyk S. Application of gas chromatography/ tandem quadrupole mass spectrometry to the multiresidue analysis of pesticides in green leafy vegetables. Rapid Commun. Mass Spectrom., 22 (2008) 3791–3801. Wan H.B., Wong M.K., Lim P.Y., Mok C.Y. Small-scale multi-residue method for the determination of organochlorine and pyrethroid pesticides in vegetables. J. Chromatogr. A., 662 (1994) 147–152. Windal I., Hanot V., Marchi J., Huysmans G., Overmeire I.V., Waegeneers N., Goeyens L. PCB and organochlorine pesticides in home-produced eggs in Belgium.

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Science of the Total Environment, 407 (2009) 4430–4437. Xiaoyn Ye, Bishop A.M., Needham L.L., Calafat A.M.. Automated on-line column-switching HPLC-MS/MS method with peak focusing of measuring parabens, triclosan, and other environmental phenols in human milk. Anal. Chim. Acta., 622 (2008) 150–156. Yang M., Park M.S., Lee H.S. Endocrine disrupting chemicals: Human exposure and health risks. J. Environ. Sci. Health Part C, 24 (2006) 183– 224. Yenisoy-Karakas S. Validation and uncertainty assessment of rapid extraction and clean-up methods for the determination of 16 organochlorine pesticide residues in vegetables. Ana. Chim. Acta, 571 (2006) 298– 307. Zhang H., Chen Z., Yang G., Wang W., Li X., Li R., Wu Y. Microwave pretreatment and gas chromatography-mass spectrometry determination of herbicide residues in onion. Food Chem., 108 (2008) 322–328.

Chapter 5 Pesticides: Herbicides and Fungicides Iván P. Román Falcó, Lorena Vidal, and Antonio Canals

Introduction The term pesticide refers to any substance used to control something that has been designated as pest, and that covers a diverse and large number of organisms. As a result, there are many different types of pesticides, ranging from rat poison and slug pellets to a wide variety of chemicals used to control insects, weeds, and fungi. Without the use of pesticides, worldwide fruit and vegetable production would significantly fall [1]. However, the consequences of their use and the realization that some foods contain residues of these compounds are of paramount importance to the human health. A classification (major groups and categories of products) of pesticides is as follows [2]: • Fungicides and bactericides • inorganic fungicides • based on carbamates and dithiocarbamates • based on benzimidazoles • based on imidazoles and triazoles • based on morpholines • other fungicides • Herbicides, haulm destructors, and moss killers • based on phenoxy-phytohormones • based on triazines and triazinones • based on amides and anilides • based on carbamates and bis-carbamates Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

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• based on dinitroaniline derivatives • based on derivatives of urea, uracil, or sulphonylurea • other herbicides Insecticides and acaricides • based on pyrethroids • based on chlorinated hydrocarbons • based on carbamates and oximecarbamate • based on organophosphates • based on biological and botanical products • other insecticides Molluscicides Plant growth regulators • physiological plant growth regulator • antisprouting products • other plant growth regulators Other plant protection products • mineral oils • vegetal oils • soil sterilants (including nematicides) • rodenticides • all other plant protection products

Fungicides are chemical compounds or biological organisms used to kill or inhibit fungi or fungal spores. Fungi can cause serious damage in agriculture, resulting in critical losses of yield, quality, and profit. Fungicides are used both in agriculture and to fight fungal infections in animals. Chemicals used to control oomycetes, which are not fungi, are also referred to as fungicides because oomycetes use the same mechanisms 127

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as fungi to infect plants. Fungicides can either be contact or systemic substances. A contact fungicide kills fungi by direct contact; a systemic fungicide must be absorbed by the affected organism. Most fungicides that can be bought at retail are sold in a liquid form. The most common active ingredient is sulfur, present at 0.08% in weaker concentrates and as high as 0.5% for more potent fungicides. Fungicides in powdered form are usually around 90% sulfur and are very toxic. Other active ingredients in fungicides include neem oil, rosemary oil, jojoba oil, and the bacterium Bacillus subtilis. Fungicide residues have been found on food for human consumption, mostly from postharvest treatments. Some fungicides are dangerous to human health, such as vinclozolin, which has now been removed from use [3]. The Fungicide Resistance Action Committee (FRAC) has published an exhaustive list of commercial fungicides according to their mode of action and resistance risk [4]. An herbicide is a substance used to kill unwanted plants. Selective herbicides kill specific targets while leaving the desired crop relatively unharmed. Some herbicides act by interfering with the growth of the weed and are often synthetic imitations of plant hormones. Herbicides used to clear waste ground, industrial sites, railways, and railway embankments are nonselective and kill all plant material with which they come into contact. Smaller quantities are used in forestry, pasture systems, and management of areas set aside as wildlife habitat [5]. For fruits and vegetables, the require ments for residue analysis for herbicides and fungicides, as well as pesticides, are established by organizations such as the World Health Organization (WHO), the Japanese Food Chemical Research Foundation, EEC Directives, and the U.S. Environmental Protection Agency (US-EPA) [6–11]. These organizations establish for which herbicides and fungicides (pesticides) residue level testing must occur in different produce,

and they establish the maximum residue limits (MRLs) allowable. However, there is no global regulation, and therefore a barrier to international trade is often produced due to the distortion of the conditions of competition in the market [12]. In an attempt to facilitate world trade while protecting the health of the consumers, the Food and Agriculture Organization (FAO) and WHO have established a joint Codex Alimentarius Commission to coordinate food standards, one of which is a universal MRLs [13]. A variety of natural compounds and anthropogenic chemicals are known to influence the endocrine system, thus they are called endocrine disruptors (EDs) or endocrine-disrupting compounds (EDCs). Endocrine-disrupting effect is obtained by mimicking the action of the steroid hormones and has been associated with several reproductive disorders as well as cancerogenesis both in animals and human beings. Studies have given rise to concerns that lowlevel exposure to EDCs might cause adverse effects in wildlife and humans. Therefore, EDCs are of concern due to their ecotoxicological and toxicological potencies. Some examples of EDCs are natural estrogens (e.g., 17β-sitosterol, estrone), natural androgens (e.g., testosterone), phystosteroids (e.g., 17β-sitosterol), isoflavenoids (e.g., daidzeine), synthetic estrogens (e.g., 17βethinylestradiol), phatalates, alkylphenol ethoxylate surfactants, dioxins, coplanar polychlorinated biphenyls (PCBs), parabenes (hydroxybenzoate derivates), bisphenol A, organotins, and several pesticides (most prominent being organochlorine insecticides such as endosulfan or DDT; herbicides such as atrazine, linuron, simazide, glyphosate, trifluralin, or diuron; fungicides such as benomyl, vinclozolin, or carbendazim and its derivatives). Because these compounds are fat soluble, it is likely they are accumulating from the environment in the fatty tissue of animals we eat; therefore, food is a major source of pollutant exposure.

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New trends in herbicide and fungicide analysis Over the last few years, pesticide analysis, and more specifically herbicides and fungicides, in food has become a hot topic for analytical chemists. This is mainly a consequence of the following [14]: • Implementation of new and more restrictive regulations for these active substances and their authorized MRLs • Social perception and consumer trends regarding the presence of chemicals in food • Development of new reduced-risk compounds by manufacturers • Development and introduction of abundant new and more powerful analytical instrumentation in food-control laboratories. In addition, recently formulated herbicides and fungicides are quite different from their predecessors in their physical properties. Most of these new herbicides and fungicides are smaller in molecular weight and were designated to break down rapidly in the environment. To successfully identify and quantify these compounds in food, a new analytical scenario has, therefore, been defined in which more careful consideration must be given to the application of improved analytical methods (i.e., sample preparation and modern instrumental techniques for identification and quantification). There are a large number of complex matrices and combinations of analytes, making rather complicated the definition of a starting point for the measure. Even once this step is determined, the choice of extraction/ separation/cleanup technique(s) and detection technique applicable to our purposes is not easy. For instance, the procedure employed for sample preparation and quantification of food samples is dictated by the basic composition of the food matrix, especially the fat content. The perfect extraction technique is sometimes an impossible ideal due to the wide range of analyte properties,

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such as the octanol-water partition coefficient (log Kow from below −4 to over 5), water solubility, and so forth. Even finding reference material to check the performance of the developed method could be an arduous task, thus the analyst may have to resort to a spiked sample. Hence, analysis of herbicides and fungicides is quite a difficult task from beginning to end. Previously, an excellent revision of conventional sample preparation (i.e., extraction, cleanup, derivatization, and chromatographic separations) and detection techniques of fungicide and herbicide residue analysis in food was published [15]. This chapter, therefore, addresses new trends in herbicide and fungicide residue analysis that have occurred since that publication.

Sample preparation Solid–liquid extraction, liquid–liquid extraction, and solid–phase extraction Regarding the solvent extraction techniques, solid-liquid extraction or liquid-liquid extraction is used for solid or liquid samples, respectively. Herbicide/fungicide extraction from solid samples is quite difficult; therefore, apart from conventional solid-liquid extraction, assisted extraction such as ultrasounds, microwaves, pressurized liquid extraction, supercritical fluid extraction, and so on are attracting the interest of analysts [16–21]. Liquid-liquid extraction (LLE) and solidphase extraction (SPE) [22] are the most commonly used extraction techniques in food analysis of herbicides and fungicides. However, these techniques present many disadvantages: they use large amounts of potentially toxic and normally expensive organic solvents, they are time consuming, and samples require manipulation (in the case of LLE). On the other hand, SPE employs expensive materials, is time consuming, and usually has carryover effects. For these reasons,

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miniaturization of the LLE equipment attempts to eliminate or minimize these drawbacks. Four different liquid microextraction techniques have been used, such as supramolecular solvent-based microextraction (SUSME) to determine benzimidazolic fungicides (carbendazim, thiabendazole, and fuberidazole) in fruits and vegetables [23]; dispersive liquid-liquid microextraction (DLLME) to determine captan, folpet, and captafol in apples [24]; hollow fiber supported liquid membrane extraction (HFSLME) to determine carbendazim and cyprodinil in cucumber, tomato, and pepper; liquid-liquidliquid microextraction with a hollow fiber membrane (HF-LLLME) to analyze phenylurea herbicides in vegetables and phenoxy herbicides in bovine milk [25, 26]; and membrane-assisted solvent extraction (MASE) to determine hymexazole, drazoxolon, vinclozolin, chlozolinate, oxadixyl, and famoxadone in wine, must, and fruit juices [27]. In the case of SPE, the most important improvement has been the introduction of molecular imprinted polymers (MIPs) as sorbent phase. They can be found in SPE columns for extraction of pyrimethanil, cyprodinil, and mepanipyrim from wine [28]; phenylurea herbicides in carrot, potato, corn, and pea samples [29]; and phenoxy acid herbicides in honey [30]. MIPs have also been used in solid-phase microextraction (SPME) for the determination of triazines (i.e., simazine, propazine, desisopropyl-atrazine, atrazine, tert-butylazine, desethylatrazine, and cyanazine) from environmental and food samples [31]. Matrix solid-phase dispersion Matrix solid-phase dispersion (MSPD) is a patented process developed in 1989 by Barker for the disruption and extraction of highly viscous, solid and semisolid samples [32]. MSPD has been applied to the extraction of drugs, pesticides, and other pollutants from animal tissues, vegetables, and fruits. An

excellent review by the pioneer of the technique is recommended for a more thorough understanding of the topic [33]. This topic has been recently reviewed by García-López et al. [34], as well. Briefly, the main steps can be summarized as the following: 1.

2.

3.

4.

5.

A relatively small sample is blended with a mortar and pestle using a bonded-phase solid support (usually 1:4 sample-tosupport ratio, for example 0.5 g sample and 2 g C18- or C8-bounded silica material) and transferred to a syringe barrel with a frit in the bottom. Viscous samples (such as milk) should be placed in a test tube or a syringe barrel and mixed with a spatula or similar device. A second frit is placed above the sample, and the sample is pressed by a modified syringe plunger. The solvent is then loaded and the analytes eluted by gravity or vacuum (usually 8 mL; most of the analytes are eluted in the first 4 mL). The collected eluates are solvent reduced or evaporated to dryness and reconstituted with the solvent of choice. The extract can be submitted to further extraction techniques or chromatographic analysis.

A certain degree of selective fractionation is possible by using solvents of different polarities, mixtures of solvents, or solvents modified by acids, bases, or salts. Macromolecular components or interferences from the sample matrix can be removed in this way. This technique is similar in certain aspects to dispersive solid-phase extraction (DSPE) (see below) [35,36] for cleanup used together with a solvent partitioning step described by Anastassiades et al. in 2003 for the determination of pesticide residues in fruit and vegetable samples [37]. The sample size used in MSPD is limited due to the high cost of the sorbents, leading to concern about sample

Pesticides: Herbicides and Fungicides

representation and homogeneity. On the other hand, DSPE provides by the extraction process a homogenous aliquot from an original sample of any size, allowing reduced amounts of sorbent to be used. In DSPE, the sorbent is added to an aliquot of the extract rather than to the original sample, as in MSPD. Moreover, some authors pointed out that the analyte recoveries from spiked samples were sometimes low and variable for the determination of sulfonamides with some extraction techniques such as liquid–liquid partitioning, SPE, and MSPD [38], although the performance of both techniques was not compared. MSPD is prone to adapt smart modifications in order to improve the extraction efficiency, decrease relative standard deviation (RSD), and reduce sample treatment. For instance, the conventional MSPD has been compared to two ultrasound-assisted MSPDs performed with an ultrasound bath and a sonoreactor. The best results were obtained with 1-min sonication using the sonoreactor at 50%, achieving the aforementioned improvements [19]. QuEChERS QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) is a DSPE technique for extracting multiresidues of pesticides from food (i.e., fruits and vegetables). It was developed by Anastassiades et al. [37], and it has become widely used in food safety analysis [39]. The advantages of this method are speed, ruggedness, effectiveness, ease of handling, minimal solvent requirement, and less expensive to perform when compared with conventional solid-phase extraction techniques. The classical QuEChERS procedure is usually a two-stage process: sample extraction, followed by DSPE. In the sample extraction stage, the food sample is homogenized to maximize the available surface area of the sample for better extraction efficiencies. The

131

homogenized sample is placed in the extraction tube containing magnesium sulfate and sodium acetate or sodium chloride. Magnesium sulfate ensures that upon addition of acetonitrile a phase separation is induced between water and organic solvent, with the pesticides of interest being extracted into the organic phase. When acetonitrile is poured into the extraction tube containing the homogenized sample, an exothermic reaction occurs between the magnesium sulfate and water, which can lead to low recoveries of target compounds. This effect can be reduced by adding the salt and sample to the extraction tube while it is immersed in an ice bath or by weighing the sample into a fluorinated ethylene propylene (FEP) extraction tube and then adding the solvent and salts slowly. The tube is then capped, shaken vigorously, and centrifuged. The second stage of the QuEChERS method uses DSPE, which involves transferring a portion of the acetonitrile extract to a cleanup tube containing a combination of sorbents for removal of unwanted sample components. This may be followed by solvent exchange to improve compatibility of samples for GC analysis and an additional sample cleanup to reduce matrix effects, thereby improving method robustness [40,41]. Some modifications on the classical QuEChERS have been suggested [42], among which a solvent exchange was used instead of DSPE [43]. A new and simplified QuEChERS modification has been recently published as AOAC (Association of Official Agricultural Chemists) method 2007.01 to determine pesticides in fruits, vegetables, grains, and herbs [44]. The sample preparation is simplified by using a single-step buffered acetonitrile extraction and liquid–liquid partitioning from water in the sample by salting out with sodium acetate and magnesium sulfate. QuEChERS has also been used to eliminate the cleanup step before the fast determination of phenoxy acid herbicides in carrots and apples using LC/ MS/MS [45].

132

Analysis of Endocrine Disrupting Compounds in Food

Dynamic separation methods (chromatography and capillary electrophoresis) The most important and widely used separation technique for determining the herbicides and fungicides in food samples is liquid chromatography (LC), employing reversedphase materials. In general, C18 and C8 can be considered as the standard reversed-phase separation material. Chromatographic separation of ionic compounds can be achieved by different retention mechanisms, mainly ionexchange and ion-pair reversed-phase liquid chromatography (IP-RPLC). One important modification in liquid chromatography has been the use of MIPs as a stationary phase to determine thiabendazole in oranges, grapes, lemons, and strawberries [46] and to screen phenylurea herbicides in vegetable samples [47]. Due to the existence of numerous applications recently published, some revisions can be found in the literature [48–51]. Detailed information is presented in Tables 5.1 and 5.2. Gas chromatography (GC) has been one of the most commonly used techniques for separation due to its high potential for separation of herbicides and fungicides and the possibility of using a wide range of detectors. Recently, however, chromatography has become a more powerful and faster separation technique, given the new name, fast gas chromatography [52]. The primary objective of GC separation is to achieve the desired resolution of compounds of a given mixture in the shortest possible time. Different routes have been followed to develop fast gas chromatography [52]: reduction of the column length; use of above-optimum carrier gas velocity; higher isothermal temperature; higher initial/final temperature and higher temperature-programming rates (low thermal mass, or LTM) or conversion of isothermal GC to temperature-programmed GC; pressure/flow programming (low pres-

sure, LP); use of columns with lower film thickness; use of a more selective stationary phase or application of coupled columns; use of two-dimensional (2D) GC or detection (by predominant use of MS detection); and by reduction of column inner diameter (I.D.), use of hydrogen as carrier gas, and application of vacuum-outlet conditions. A comprehensive two-dimensional gas chromatography-rapid scanning quadrupole mass spectrometric (GCxGC-qMS) methodology has been developed for the analysis of trace amounts of pesticides contained in very complex real-world samples (i.e., red grapefruit extract) [53]. Similar GCxGC associated with nitrogen-phosphorus detection (NPD) has been investigated for the separation and quantification of fungicides in vegetable samples [54]. Sherma [55,56] has published two reviews on applications of thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) for the separation and quantification of pesticides in a variety of food and crop samples. Specific applications of carbendazim, thiram, thiophenate-Me, captan, and folpet in fruits and vegetables have been also published [57]. Tadeo et al. [15] described capillary electrophoresis (CE) as an alternative technique for the determination of polar compounds. Nevertheless, it was not suggested because it lacks the flexibility of chromatography in adjusting separation factors and yielded inconsistent migration times and high standard deviation. But, over the last 8 years, this has turned around, and CE and micellar electrokinetic chromatography (MEKC) have been used in many methods, and thus various reviews can be found in the literature [58–60]. Capillary electrophoresis-mass spectrometry (CE-MS) has been used for determination of thiabendazole and procymidone in fruits and vegetables [61] as well as the group of triazolopyrimidine sulfonanilide herbicides in soy milk samples [62]. Nonaqueous CE (NACE) has been

133

Ametryn, atrazin, atraton, prometon, prometryne, propazine, simetryn, terbutryne, terbuthylazine, terbutryn

2,4-Dichlorobenzoic acid, 2,4-dichlorophenoxyacetic acid, 2-(2,4,5-trichlorophenoxy) propionic acid (fenoprop), 2-(4-chloro-phenoxy)-2methylpropionic acid (mecoprop), 3,5-dichlorobenzoic acid Chlorbromuron, chlorotoluron, fenuron, isoproturon, linuron, metobromuron, metoxuron

Bovine milk

Carrot, potato, corn, pea

MIP-SPE

HF-LLLME

UA-MSPD

USE

ASE-SEC

Chlorotoluron, diuron, isoproturon, linuron, methabenzthiazuron, monolinuron, monuron Ametryn, atrazine, prometryn, propazine, simazine, terbutryn.

Extraction Technique PLE

Compounds Thiobencarb

Apple, pear, and apricot

Potatoes

Komatsuna, spinach, qing geng cai, broccoli, cauliflower, green pepper, and okra Orange

Matrix

HPLC: Kromasil 5 ODS (150 mm–4.6 mm I.D.)

HPLC: column (250 × 2 mm I.D.) packing material Inertsil-ODS-2.

GC-MS: Column: BPX5 30 m × 0.25 mm I.D., 0.25 μm film thickness.

CE in nonaqueous media

HPLC: Spherisorb S5 ODS2 C18 (2.1 × 150 mm)

GC: SGE BPX-5 Column (30 m × 0.25 mm I.D., df film thickness 0.25 μm)

Separation Technique

Figures of Merit and Remarks

Linearity: r2 ranged: 0.980–0.989; Recoveries: 93–116%, RSDs: 11–17%, LODs: 1.7–4.0 Extraction method selected: UA-MSPD with sonoreactor. Mean recovery: 83–118%. Reproducibility: RSD 8–24%, LODs: 3.8–35 μg/kg (for terbuthylazine-atraton, respectively). Linearity: 1–200 μg/L (r > 0.9965) LODs: 0.5 μg/L. Enrichment factor: 261–952 (for 2,4-DCBA and Fenoprop, respectively). RSDs: 4.56–7.02% (for 2,4-DCBA and fenoprop, respectively). Recovery: 70.8–77.0% (at 5 μg/L) and 84.0–89.5% (at 10 μg/L) Recoveries: ranged 84–106%. Procedure allowed screening at EU MRL

UV-Vis: λ = 214 nm

UV-Vis: λ = 244 nm

Detector: UV λ = 240 nm. Injection

MS: EI 70 eV Full scan (m/z 50–550)

Matrix effect study

Recovery: 84–96%, RSDs: 7.5–21%, LOD: 4 μg/kg

ESI-MS-MS: MRM mode

MS: SIM

Detection Technique

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis.

(continued)

[29]

[26]

[19]

[18]

[17]

[16]

References

134

Barley

Multi-residue (43-Herbicides)

Atrazine, cyanazide, desethylatrazine, desisopropylatrazine, propazine, simazine, terbutylazine Ametryn, atrazine, prometryn, propazine, prometon, simazine

Potato and pea

Luffa, berries, small fruit, pulses, tower gourd, grape, broad bean

Compounds 2,4-D, 2,4,5-T, 2,4,5-TP

Matrix Honey

QuEChERS

QuEChERS

MIP-SPME

Extraction Technique MIP-SPE

TOF-MS; ESI-MS-MS

MS: single quadrupole source, ESI; ion polarity, positive

LC: Symmetry-C18 column (150 × 2.1 mm I.D., 5 μm)

GC-TOF-MS; LC-ESI-MS-MS

DAD λ = 220 nm

Detection Technique DAD λ = 244 nm. MS2: ESI negativecapillary voltage

HPLC: Kromasil 5 ODS column (150 mm × 4.6 mm I.D.)

Separation Technique HPLC: Column Luna PFP(2) 150 × 4.6 mm, 3 μm.

Linearity: 1–200 μg/L (r2 > 0.99). Recoveries: 80–110% (except for Simazine 73–79%) RSDs <10%. LODs: 0.05–0.2 μg/L LOQs: 0.1–1 μg/L GC-TOF-MS: (16 herbicides) linearity coefficient ranged 97.1–98.8. LODs: ranged 1.0–2.3 μg/kg. LC-ESI-MS2: linearity: ranged 94.1–99.8 (except Pyridate 70.5). LODs: ranged 0.2–3.3 μg/kg (except Pyridate 23.2). Recoveries: ranged: 60–70% (some exceptions), RSDs: <30% (some exceptions)

Figures of Merit and Remarks HPLC-DAD: Linerarity: 7.5–75 ng/g (r2 > 0.991). LODs: 1–3 ng/g. RSD%: 13–10% (at 12.5 ng/g) and 7–12 ng/g (at 50 ng/g). HPLC-MS2: Linearity: 7.5–75 ng/g (r2 > 0.993). LODs: 0.1–1.1 ng/g. RSD%: 12–22% (at 12.5 ng/g) and 12–18 ng/g (at 50 ng/g). Recoveries: HPLC-DAD 88–112%; LC-MS2: 88–104% Recoveries: 12–25%. Repeatability: RSDs: 4–10%, interday repeatability: 7–15%

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

[39]

[36]

[31]

References [30]

135

Rice

Extraction Technique

Acetochlor, alachlor, butachlor

—

SPE

Diuron, isoproturon, linuron, metobromuron

Apple juice, rice, milk, sausage

PLE-SPE clean up: Oasis MCX sorbent.

Ametryn, atrazine, deethylatrazine, prometryn, propazine, simazine, terbutryn

Potato, carrot, lettuce, zucchini, runnerbeans, oranges, and wheat Soy shoots and soy milk

SPE

Cloransulam-methyl, metosulam, flumetsulam, florasulam, diclosulam

—

QuEChERS

Soy milk

Potato, corn, and pea

Compounds 2,4-D, 2,4-DB, 2,4,5-T, acifluorfen, clopyralid, dicamba, fluroxypyr, MCPA, MCPB, MCPP, picloram, quinclorac, triclopyr. Chlortoluron, fenuron, isoproturon, linuron, metoxuron, metobromuron

Matrix

Separation Technique

CE-UV: 100 μm I.D., 375 μm O.D., silica fused capillary packed with LiChrospher 100 RP18 5 μm particles. Capillary total length 32 cm. —

HPLC: MIP-silica particles packed in HPLC stainless steel columns (125 mm × 4.6 mm I.D.). NSM-CE-MS: CE: Detection length to the UV-detector 20 cm and total length was 87 (corresponding to the MS detection length). Injections: anodic end N2 pressure of 0.5 psi for 100 s. Separation temp 22°C and +25 kV. NACE HPLC

UPLC: column Acquity BEH C18 (2.1 mm × 100 mm, 1.7 μm film thickness).

Immunosensor

Analytical working ranges 0.05–4.0 μg/L (acetochlor and alachlor), 0.005–2.0 μg /L (butachlor). RSD: 1–10%. LODs: 0.02 μg /L (acetochlor and alachlor), 0.002 μg /L (butachlor).

Linearity: 2.5–50 mg/L (r2 ranged 0.9974–0.9997). RSD (intraday n = 9): 2.54–7.91%. RSD (interday, n = 4) 3.40–6.87%. LODs: 0.05 mg/Kg

Linearity: 25–250 μg/kg. Recoveries: 87–108%, RSDs: 7–17% LODs: 10–14 μg/kg.

UV-Vis: λ = 214 nm; λ = 200–300 nm

UV-Vis: λ = 245 nm

[62]

LODs: 74 μg/L RSD: 3.8–6.4%. RSD (interday): 6.5–8.1%.

MS: orthogonal ESI Negative ion mode. 300–450 m/z range.

(continued)

[71]

[64]

[63]

[47]

[42]

References

Linearity: range 0.5–10 mg/L of each pesticide (r2 > 0.998).

UV-Vis

Figures of Merit and Remarks LODs: 0.5–5 μg/kg Recoveries: 45–104% with RSDs: <13.3%

Detection Technique ESI–MS/MS

136 SLE-derivatization — QuEChERS

CdTe Quantum dots

Starane (fluroxypyr) Simazine Ametryn, atrazine, terbutylazine,

2,4-Dichlorophenoxyacetic acid

Wheat and maize grains and flours Orange juice, milk Orange, lemon, apple, green pepper, persimmon, grapefruit, tomato, pear, and grape n. a.

SLE-derivatization

Bromoxynil

Wheat and maize grains and flours

MSPD

Propham and maleic hydrazide

—

Nitralin and oryzalin

Potatoes

—

Paraquat

—

Extraction Technique —

Orange, lemon, apple, potato, sugar cane Grape juices, potato, and soybean

Compounds

Paraquat

Atrazine

Apple and potato

Matrix Wine grapes and other foodstuff products

Separation Technique

—

DESI compared with LC-MS

—

FIA

HPLC-UV used as reference method FIA

—

—

—

—

Competitive fluoroimmunoassay based biosensor

DESI-MS or DESI MS-MS

μ-ISLMA

UV-Vis

UV-Vis

DPV

AdSV at a hanging mercury drop electrode

MSWV

SWV at a carbon paste electrode modified with fluoroapatite

Detection Technique Single frequency impedimetric immunosensor

LODs: 250 ng/L.

Linearity: r = 0.999 LODs: 2.47 10−8 mol/L and 1.5 10−8 mol/L. RSDs: 1.14%, 0.998, 1.48% Recoveries: 98% (propham), 68% (maleic hydrazide). Linearity: 10–5000 ppm. RSDs: 0.32% LODs: 9 ppb LOQs: 30 ppb. Linearity: r = 1.00 LODs: 0.05 mg/L LOQs: 0.17 mg/L LODs: 0.1 ng/L. RSDs: 3.35%. LODs: 1–2 μg/kg in tomato and orange. RSD <15%

Figures of Merit and Remarks LOD: 8.34 ± 1.37 ppb LODs < MRL required by EC for atrazine in wine grapes and other foodstuff products Linearity: 5·10–8 -7·10– 5 mol/L (r2 0.9841 and 0.9823). RSDs: 2.16% and 2.70%. LODs: 3.5·10–9 and 7.4·10–9 LODs: 0.044 μg /L and 0.146 μg /L.

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

[96]

[95]

[94]

[92]

[91]

[84]

[82]

[81]

[80]

References [72]

137

2,4-D Cafenstrole and its metabolite (CHM-03) residues

Acetachlor, ametryn, atrazine, butachlor, chlorbufam, diclobenil, hexazinone, mefenacet, metribuzin, prometryne, propyzamid, terbumeton, trifluralin

4-Bromoaniline, 4-chloroaniline, 3,4-dichloroaniline, linuron, metobromuron, monolinuron 2-Hydroxyterbuthylazine, amidosulfuron, atrazine, chlorotoluron, isoxaflutole, linuron, metosulam, rimsulfuron, simazine.

Water

Brown rice grains

Chinese teas

Carrots, potatoes, onions

Honey

Compounds Atrazine

Red wine

Matrix

Extraction Technique

Separation Technique

GC: BP10 (30 m × 0.25 mm × 0.25 μm 14% cyanopropylphenyl + 86% dimethylpolysiloxane) column LC-MS: Column Polaris C18-A HPLC column (150 × 2.0 mm I.D., 3 μm, 200 A).

SPME: 85 μm PA fiber

On-column-LLE

GC: HP-5 MS column (30 m × 0.20 mm I.D. and 0.25 μm film thickness)

HPLC: RP-C18 Chromolith performance column (100 × 4.6 mm, I.D.)

—

—

LLE-GPC-SPE

SLE-LLE-SPE

—

SPE

Detection Technique

qMS: positive ESI.

NPD Q-MS: SIM

MS: scan/SIM mode. Electron energy: 70 eV

Impedimetric immunosensor based on interdigitated microelectrodes Surface plasmon resonance immunosensor MS: SIM

RSD: 8–27%. Recoveries: 71% and 90%

(continued)

[102]

[101]

[100]

[99]

[98]

[97]

Determination range: 10 ppt–800 ppb, LODs: 10 ng/L Recoveries: cafenstrole: 87.0–92.5 and 87.6– 88.3%% grains and straw of brown rice. Cafenstrole metabolite: 81.5 to 81.6% and from 76.1 to 78.5% RSDs: 1.4–6.6%. LODs: 0.002 and 0.02 ppm and 0.025 and 0.04 ppm, respectively Linear determination of coefficients (r2) higher than 0.990, except for 4-t-pentylphenol (r2 > 0.910) and Mefenacet (r2 > 0.905). Recoveries of the analytes ranged from 60.7 to 136.7%. Repeatability RSD: 3.0– 30.8%. LOQ: 0.0099 (for hexachlorobenzene) μg/mL and 2.45 μg /mL for propargite Linearity: r > 0.995. Recoveries: 83–92% RSDs: 4–8% LOQs: 0.8–2.2 μg/kg (NPD) LOQs: 0.1–0.7 μg/kg (MS)

References

Figures of Merit and Remarks LODs: 0.19 μg/L in red wine after SPE

138

Grape, pear, apple, lemon

QuEChERS

LLE-SPE: Oasis HLB SPE cartridge

Atrazine, chlorsulfuron, diuron, isoproturon, haloxyfop-methyl

Multiresidue (24-herbicides)

SLE

Chlorpropham

SLE

Atrazine, bromozynil, chlorotoluron, diuron, flufenacet, pendimethalin, simazine

Frozen and canned vegetables, canned fruit, and ready-toeat salads: tomato, peas, lettuce, apple puree Lemon and raisin

—

Alachlor, atrazine, dinoseb, isoproturon, metolachlor, metolcarb, simazine

Apple, cranberry, grape, and orange juices as well as fruit peel and salad leaves Strawberry

Extraction Technique SPE: HLB cartridges (200 mg) from Oasis

Compounds Multiresidue (24-herbicides)

Matrix Fruit-based soft drinks

LC: Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm I.D., 5 μm particle size)

TOF-MS: EI-positive ion mode MS-MS: hybrid triple quadrupole/ linear IT

MS-MS: orthogonal Z-Spray-ESI

Linearity: 10–500 μg/L. Recoveries: 71–108%. (except Haloxyfop-methyl in lemon 45–51%) RSDs: <23%. LODs: <0.01 mg/kg LOQs: 0.01 mg/kg Linearity: r2 > 0.997. LODs: 0.01–5 μg/kg (MS-MS) LODs: 0.06–15 (TOF-MS)

ESI(+)-MS-MS selected reaction monitoring. SRM

LC: Xterra MS C18 column (2.1 mm × 150 mm, 3.5 μm particle size)

UPLC: Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm particle size)

Calibration: matrix-matched standards (0.005–0.04 mg/ kg) r2 = 0.99 Recoveries: 81.0–103.2% RSDs: 3.0–19.6% Linearity: 0.5–100 μg/L (r2 ≥ 0.99). Recoveries: 65–94.4% with RSDs: 6.1–19.0% at 10 μg/kg. LODs: 2–7 μg/kg

MS-MS: MRM TOF-MS: accurate mass

UPLC: Acquity UPLC BEH C18 column (50 mm × 2.1 mm I.D., 1.7-μm particle size)

Figures of Merit and Remarks Recovery rates between 70% and110%. Accurate mass. LODs: 0.01–2 ng. RSDs: around 20%

Detection Technique TOF-MS: ESI positive ion mode Screening with APGD-MS

Separation Technique LC: Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μm particle size) —

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

[108]

[107]

[106]

[105]

[104]

References [103]

139

Rice, maize, and onion

Ametryn, atrazine, cyanazine, prometon, prometryne, propazine, secbumeton, simazine, simetryn, terbuthylazine 1,3,5-Triazine, ametryn, atrazine, cyanazine, prometryn, propazine, simazine, terbutryn MIP-SPME

SFE

SLE-SPE

Chlorpropham

Eggs

—

Simazine

Cucumber, potato, tomato, fresh cucumber juice, unskimmed milk Potatoes

LLE Vs PLE

Extraction Technique

Propanil

Compounds

Apricot, cherry, orange, peach, pear, strawberry

Matrix

GC: CP-Sil 8 CB column (50m × 0.25 mm I.D., 0.12 μm film thickness) GC: CP-Sil5-CB (30m × 0.25 mm I.D.)

GC: Rtx-50 (50% diphenyl/50% dimethylpolysiloxane stationary phase, 30 m × 0.25 mm I. D., 0.25 μm film thickness). Rtx-1 GC: DB-17 (30 m × 0.25 mm, 0.25 μm film)

—

LC: Luna C18 (150 mm × 4.6 mm I.D., 5 μm)

Separation Technique

FID MS

FTD

ECD TSD

Potentiometric immunosensor

Triple quadrupole (QqQ) quadrupole ion trap (QIT) quadrupole-time-offlight (QqTOF)

Detection Technique

[113]

Linearity: r2 > 0.9925 Absolute recoveries: 3.4–9.6%. LODs: 14–95 μg /L (FID). LODs: 10–90 μg /kg (onion and maize) LODs: 30–230 μg /kg (rice). Recoveries: 85–96.7%. RSDs: 5.56–10.8%.

(continued)

[112]

[111]

Recovery averaged: atrazine: 90.4% with an RSD of 3.3% Others >73.4%.

Linearity: r2 > 0.99 Recoveries: ECD: 73–101% with RSDs: 4–15%; TSD: 86–120% with RSDs: 3–20%.

[109]

Linearity: 1–1000 μg/kg (QqQ), 20–5000 μg/kg (QIT) and 70–7000 μg/kg (QqTOF). Repeatabilities (RSDs): QqQ (5–12%); QIT (6–15%); QqTOF (14–19%) Recoveries 88–92% (PLE), 64–77% (LLE) LODs: 1 μg /kg (QqQ), 20 μg / kg (QIT), 70 (QTOF). LOD: 3 μg/L Compared with ELISA

[110]

References

Figures of Merit and Remarks

140 SLE LLE-SPE

Atrazine, simazine, prometryn Atrazine, simazine, prometryn

Atrazine, diuron, simazine, terbuthylazine. Atrazine, diuron, simazine, trebuthylazine

Fresh and processed olives Olive oil

Olive oil and olives

Olive oil

LLE-MSPD

GPC

—

Diclofop-acid and diclofop-methyl

Herbicides

Extraction Technique SFE

Compounds Paraquat and diquat

Matrix Olive oil

LC and LCxLC: System A: C18-Luna column (150 × 4.60 mm, 5 μm). System B: ChiralAGP column (100 × 30 mm, 5 μm). GC: Trx-1701 column (30 m × 0.25 mm, df film thickness0.25 μm). GC-NPD: Zebron ZB-1 (30 m × 0.32 mm I.D., df film thickness 1 μm) GC-ECD: Zebron ZB-5 (30 m × 0.25 mm I.D., df film thickness 0.25 μm). GC-MS/MS: CP-SIL 5CB (30 m × 0.25 mm I.D., df film thickness 0.25 μm). LC-TOF-MS: Zorbax Eclipse XDB-C8 (150 mm × 4.6 mm, 5 μm particle size)

Separation Technique HPLC: μBondapak C18 (300 mm × 3.9 mm I.D., 10 μm particle size)

TOF-MS

IT-MS-MS: EI mode

NPD, ECD

NPD

UV-Vis

Detection Technique DAD: confirmation method (HPLC) Screening method: amperometric

Recoveries: 82–136, RSDs: 5–11%, LODs: 0.1–0.5 μg/ kg. LOQs: 0.2–5.0 μg/kg. Linearity: Matrix matched std. Range: 5–500 μg/kg (using extracted ion chromatograms ([XIC]) (r > 0.99). Matrix effects: 14–36% matrix induced suppression. LODs: 1–5 μg/ kg. RSD <4%.

Linearity: 5–250 μg/L Recoveries: 96–99% RSDs: 1–11% Linearity: 20–500 μg/kg (r2 ranged: 0.9937–0.9987). RSD: 5.7–10.1%. Recovery: 68.8–85.6%. LODs: 10 μg/L.

Figures of Merit and Remarks Amperometric: Linearity: 25–1000 μg/kg. LODs: 10–11 μg /kg. RSDs: 4.1–4.9% HPLC: Linearity: 40–1000 μg /kg. LODs: 2–3 μg/kg Recoveries: 92–108% LODs: 1.25 and 1.87 mg/L for diclofop-acid; 2.70 and 3.02 mg/L for diclofopmethyl, enantiomers, respectively

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

[119]

[118]

[117]

[116]

[115]

References [114]

141

Multi-residue (16-herbicides)

Diclofop-methyl, lactofen, ethofumesate, fluroxypyrmeptyl, acetochlor

Multiresidue (31-herbicides)

4,6-Dinitro-o-cresol

Barley, oats, potato, water

Water

Bovine milk

Lemon juice

Compounds Atrazine, diuron, simazine, terbuthylazine, terbutryn, trietazine

Olive oil

Matrix

Extraction Technique

SPE-microwave derivatization

SPE

SPE

IA-SPE

LLE-GPC

Separation Technique

Detection Technique

CD: λ = 230, 254 nm.

MS2: 0.4 mL/min of column effluent to ESI-MS2. Positive/ negative modes.

UV-Vis: λ = 264 nm

HPLC: HP 5 μm C18 guard (7.5 mm × 4.6 I.D.); Column (250 mm × 4.6 I.D.).

HPLC: C18 column 5 μm (250 mm × 4.6 I.D.). For comparison with spectrophotometric methods.

UV-Vis: λ = 227. LC-MS-MS

TSD ECD MS

HPLC: column (250 mm × 4.6 mm I.D.).

HPLC: Prontosil 120–3-C18-AQ column (250 mm × 436 mm I.D., 3 μm).

GC-TSD/ECD/MS: GC: Column CP-SIL 5CB (30 m × 0.25 mm I.D., df film thickness 0.25 μm).

[120]

(continued)

[124]

[123]

[122]

[121]

References

Figures of Merit and Remarks Recoveries and RSDs (n = 10) values were 91–124% and 1–8% (GC-ECD), 82–100% and 9–20% (GC-TSD), and 89–105% and 4–14% (GC–MS/MS), respectively Recoveries: >80% in water samples. LODs ranged 20–70 ng/L (HPLC-UV) for tap water; 30–100 ng/L for surface water Linearity: r2 > 0.99; RSD <7.5%. LODs: 0.06, 0.05 mg/L for single enantiomers of ethofumesate, diclofopmethyl, respectively. Enantiomeric resolution of quiral pesticides Linearity: up to 200 ng with r2 ranged: 0.9878–09976. Recoveries: 78–104% with RSDs <13%. Accuracy: 82–120% with RSDs <11%. LOQs: 0.008–1.4 μg/L. Periodate method: Linearity: 7.5–225 mg/L. LOD: 1.6 mg/L. Cu(II)-Nc method: 0.44– 4.44 mg/L. LOD: 0.2 mg/L Recoveries >95%

142

Grapefruit, orange, blackcurrant, apple juices

Carrot, grape, and multivegetable juices

Apple and cherry juices and peach nectar

Matrix Apple juice

Alachlor, atrazine, cyanazine, EPTC, metolachlor, metribuzin, pendimethalin. prometryn, propachlor, simazine, terbumeton, terbuthylazine, terbutryn, trifluralin, triallate Atrazine

Compounds 2,4-dichlorophenoxyacetic acid, 4-chloro-2methylphenoxyacetic acid, 2-(2,4-dichlorophenoxy) propanoic acid, 2-(4-chloro2-methylphenoxy) propanoic acid, 4-(2,4-dichlorophenoxy) butanoic acid, 4-(4-chloro-2methylphenoxy) butanoic acid, 2-(2,4,5-trichlorophenoxy) propanoic acid, 2,4-dichlorophenoxyacetic-1methyl ester and 2,4-dichlorophenoxyacetic-1butyl ester Trifluralin

SLM-SPE

MSPD

SPE

Extraction Technique SPE

Linearity: 1–25 mg/L (r = 0.996). LOD: 0.3 mg/L. Preconcentration factor: 100; Recoveries(50–500 μg/L): 36.2–50.2% (orange juice); 39.8–68.5% (apple juice); 46.3–52.5% (grapefruit juice)

MS: EI 70 eV

UV-Vis: λ = 214 CE-UV: Background electrolyte: 10 mM phosphate buffer, 60 mM SDS, 20% MeOH adjusted to pH 9.2. Voltage: 20 kV; 10 s of pressure injection time.

GC: Column ZB-5MS, (30 m × 0.25 I.D., film thickness 0.25 μm)

Linearity: r > 0.9988, Recoveries: 93.8–99.5%, RSD: <3.4% (range 1–16 μg/kg) Linearity: ranged from 0.01 to 0.05 mg/L. 0.9963–0.9999. Recoveries: 82–115% RSD: <10%; LODs: 0.1–1.6 μg/L

UV-VIs: λ = 220 nm

HPLC: C18 5 μM Luna column (250 × 4.6 mm I.D.)

Figures of Merit and Remarks Linearity: 30–170 μg/kg, (2,4-D, MCPA, 2,4-D-1metyl ester), 20–120 μg/kg (for 2,4-CP, MCPP, 2c4-DB, MCPB, 2,4,5-TP); 70–170 μg/kg (for2,4-D-1butyl ester) (r ranged 0.992–0.999) LODs: 0.005–0.018 mg/kg. Recoveries: 84–99% RSD: 1–4%

Detection Technique UV-Vis: λ = 232 nm, (except 2,4-D1butyl ester 283 nm)

Separation Technique HPLC: column 3 μm Hypersil C18 BDS (150 mm × 0.3 mm I.D.)

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

[128]

[127]

[126]

References [125]

143

Atrazine, dipropetryn, prometryn, sebuthylazine, simetryn, terbuthylazine, terbutryn

Ametryn, atrazine, prometryn, propazine, simazine, simetryn, terbutryn Atrazine, desmetryn, propazine, sebuthylazine, secbumeton, simazine

Atrazine, ametryne, deethylatrazine, deisopropylatrazine, prometryne

Multiresidue (22-herbicides)

Orange juice

Tomatoes, strawberry juice, and milk

Bovine milk

Breast milk

Water

Compounds Desmetryne

Apple juice

Matrix

Extraction Technique

Direct injection

SPE

HFM-SPME

SPE with magnetic MIP beads

IA-SPE

SPE: florisil (1 g, 6 mL) and Extra-Sep C18 (1 g, 6 mL)

Separation Technique

RRLC: ZORBAX Eclipse XDBC18 (4.6 mm × 50 mm, 1.8 μm particle size)

GC-MS: column: BPX-5 SGE (25 m × 0.22 mm I.D., 0.25 μm film thickness)

HPLC-UV: Analytical column: C18 Dikma (250 mm × 4.6 mm I.D., 5 μm). GC-MS: Column: DB-5 (30 m × 0.32 I.D., 0.25 μm-film thickness).

GC-NPD: Column HP-5MS (25 m × 0.25 I.D. , 0.25-pm film thickness). Solvent vapor exit.

—

Detection Technique

MS-MS: triple quadrupole mass spectrometer fitted with an ESI MS source. MRM.

MS: SIM mode.

MS: SIM Mode. Interface temp. 200°C.

UV-Vid: λ = 225 nm

NPD: Detection: flow split 1:1 (NPD: FID)

Adsorptive stripping voltammetry

Figures of Merit and Remarks Concentration range: 2.0·10−9–1.0·10−7 mol/L LODs: 2.4·10−9 and 4.2·10−10 mol/L RSDs: 3.6–3.7%. Linearity: N. D.; LODs: 120–170 pg FID, ∼15 pg NPD. RSD (n = 6): ∼6.6–7.1%. Recoveries: 64–88% (1 μg/L, sample vol 10 mL). Waste water: 78–86%. Orange juice: 100–105% Linearity: 1.0–40.0 μg/L. LODs: 0.20–0.63 μg/L. Recoveries: 71.6–118.5%. RSD: 2.6–10.7%. Linearity: 0–200 μg/L (r ranged 0.9799–0.9965.) Recoveries: 88.4–107.2% (at 20 μg/L); 56.9–98.2% (at 1 μg/L). LODs: 0.003– 0.013 μg/L; LOQs: 0.006–0.021 μg/L Calibration. 1–100 μg/L. Linearity: r ranged 0.9964– 0.9997. LODs 0.3 μg/L. LOQs: 1 μg/L. RSD: 2–5% Accuracy: 98.63–104.62%. Recovery: 58.64–63.22% Linearity: 30–2000 ng/L (r > 0.99) LODs: <15 ng/L and correlation Precision <20% Accuracy confirmed by external evaluation

(continued)

[134]

[133]

[132]

[131]

[130]

[129]

References

144 Atrazine

Atrazine

Oxadiazon

Wine

Wine grape

Ground water, must, wine, and human urine

SPME

—

—

—

SPE

Oryzalin

Atrazine

Extraction Technique LLE-SPE

Compounds Azisulfuron, falzasulfuron and halosulfuron-methyl

Wine

Matrix Rice, corn, cotton seed, ginkgo nut, chestnut, almond, walnut, cucumber, pumpkin, orange, grapefruit, mandarin, lemon, and grape Oranges, lemons, peaches, cherries

GC-MS: GC: HP-1 (30 m × 0.25 mm I.D., 0.25 μm film thickness).

—

—

—

LC: Column: Zorbax SB-C8 (75 mm × 4.6 mm I.D., 3.5 μm particle size)

Separation Technique HPLC: LC-MS: Column packed with Tosoh TSKgel Super-ODS (100 mm × 2.0 mm I.D.).

Immunodevice based on negative dielectrophoresis Conductimetric immunosensor based on antibodies labeled with gold nanoparticles Impedimetric immunosensor using interdigitated μ-electrodes MS: EI in SIM mode

MS-MS: Electrospry negative polarity. MRM.

Detection Technique UV-Vis: λ = 245 nm; MS: ESI negative; SIM.

[136]

[138]

[139]

[140]

Linearity: r > 0.9997 (0.1–-50 μg/L). Average recoveries (fortification levels 0.01–1 μg/g): 85–89%. RSDs: 2.7–4.4%. LODs: 0.02 μg/g LOQs: 0.010 μg/g LODs: 0.11 g/L in buffer and 6.8 μg/L in pretreated wine samples. LODs: 0.1–1 μg/L

LOD: 8.34 ± 1.37 μg/L.

Linearity: 0.5–50 μg/L. LODs: < 0.02 μg/L. Reproducibility: RSDs 6.5–13.5%.

[137]

References [135]

Figures of Merit and Remarks Recoveries: ranged 77.0– 112.3. LODs: 0.01 μg/g (azimsulfuron and halosulfuron-methyl), and 0.02 μg/mL for flazasulfuron.

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

145

Chlormequat and mepiquat

Glyphosate and aminomethylphosphonic acid (AMPA)

Alachlor, acetochlor, and metolachlor and their corresponding OXA and ESA derivatives

Atrazine, desisopropylatrazine, desethylatrazane, desisopropyldesethylatrazine, hydroxyatrazine, propazine, simazine, terbutylazine. Amitrole

Wheat flour and mixed cereal flour

Soybean

Natural water, surface and groundwaters

Water

Apple

Oxadiargyl

Rice

Compounds Trifluralin, terbuthylazine

Wine

Matrix

Extraction Technique

SPE-derivatization: C18 cartridgederivatizing agent: CNBF

—

SPE

SLE

PLE

QuEChERS

SPME

Separation Technique

Detection Technique

Immunoassay with fluorescence detection UV-Vis (λ = 360 nm). IP-HPLC: hypersil ODS C18 column (4 mm × 3 mm I.D.)

Q-MS UV-Vis

ESI-MS-MS: MRM.

ESI–MS–MS: positive ionization mode; MRM

ECD

MS: EI 70 eV. SIM.

—

GC: Column: RTX-5MS 30 m × 0.25 mm I.D., 0.25 μm film thickness HPLC: Discovery column (150 × 4.6 mm)

HPLC: Zorbax Eclipse1 RDB C8 column (150 mm × 4.6 mm I.D., 5 μm particle size)

LC: Spherisorb column SCX (50 × 4.6 mm I.D., 5 μm)

GC: Zebron ZB-1 30 m × 0.32 mm, 0.25 μm

GC: HP-5-MS Column (30 m × 0–25 mm I.D., film thickness 0.20 μm.)

Figures of Merit and Remarks

Linear range: 166–415 mg/L (r2 = 0.9996). Recovery: 94.17–105.67%. RSDs: 1.57–6.44%. LOD: 0.10 mg/ kg.

Trifluralin: Linearity: r > 0.945, RSD: <16% (1 μg /L), RSD <7.5% (100 μg/L). LOD: 0.15 μg /L. Terbuthylazine: Linearity: r > 0.968, RSD: < 11% (1 μg/L), RSD: <5.9% (100 μg/L). LOD: 0.55 μg/L Recovery: 82.9–84.8. RSD: 0.2–2.7%. LODs: 0.001 mg/kg. LOQ: 0.01 mg/kg Linearity: r2 > 0.9986 Recoveries: 83–99% RSDs: <10%. LODs: <0.1 μg/g Recoveries: 73.9–109.1% RSDs: 5.3–14.5% LODs: 0.09 and 0.1 mg/kg for glyphosate, and AMPA, respectively LOQ: 0.30 and 0.34 mg/kg for glyphosate, and AMPA, respectively Recoveries: ranged 76–100% for PCs, 41–91% for OXAs, and 47–96% for ESAs. RSD of absolute recoveries <12%. LODs: 1–8 ng/L for PCs, 1–7 ng/L for OXAs, 10–90 ng/L for ESAs. Dynamic range: 0.010– 7.5 μg/L. LODs: 1.3 ± 0.9 ng/L for simazine

(continued)

[147]

[146]

[145]

[144]

[143]

[142]

[141]

References

146 Multiresidue (36-herbicides)

QuEChERS

Multiresidue (41-herbicides)

Cabbage, potato, spinach, apple, orange, rice, soybean

QuEChERS

Propham, chlorpropham, propyzamide, prometryn, diflufenican

Cherry, nectarine, orange, apricot, pineapple, French bean, cucumber, eggplant, green cabbage, potato, avocado, lemon, clementines, grape, plum, and peach Green peppers, tomatoes and oranges QuEChERS

QuEChERS

Multiresidue (31-herbicides)

Tomato, pear, orange

Extraction Technique QuEChERS

Compounds Terbuthylazine

Matrix Baby food

LC: Zorbax Eclipse SB-C18 column (150 mm × 4.6 mm, 1.8 μm particle size) HPLC: Column: Ascentis C18, 2.1 × 100 mm, 3 μm

GC: CP-Sil 8-MS column (30 m × 0.25 mm I.D., 0.25 μm film thickness) LC: Alltima C18 column (150 × 3 mm I.D., 5 μm particle size)

LC: Zorbax Eclipse XDB C8 column (150 mm × 4.6 mm, 5 μm particle size)

Separation Technique GC: CP-Sil 8 CB (15 m × 0.15 mm I.D., 0.15 μm film thickness)

API-MS: ESI.

MS-MS: ESI interface in positive ion mode. MRM

IT-MS: Full-scan (60–550 m/z) EI (automode) with 10 μA filament current MS-MS: ESI+ mode

MS-MS: Triplequadrupole with ESI source in positive mode

Detection Technique MS: (EI) mode (70 eV) in SIM mode

Recoveries: 70–120% RSDs: <20%

Linearity: over 3 orders of magnitude (r > 0.99). LODs: 0.3–50 μg/kg

Figures of Merit and Remarks Modified QuEChERS method utilizing column-based SPE cleanup instead of dispersive SPE provides lower recoveries, but slightly better LOQs. More than 95% of the pesticides under study presented recoveries between 80% and 110% representing 85%, 65%, and 78% for tomato, pear, and orange, respectively. RSDs: <20% More than 90% of the cases were below or equal to 5 μg/ kg and the most frequent LOD was 1 μg/kg. Recoveries: 90–110% for 70–80% of the analytes, RSDs: <10% for majority of analytes

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

[152]

[151]

[150]

[149]

References [148]

147

QuEChERS

QuEChERs

Multiresidue (33-herbicides)

Multiresidue (36-herbicides)

Apple, pear, tomato, potato, pepper, cucumber, olive oil Green peppers, tomatoes, cucumbers, and oranges

QuEChERS

Atrazine, butachlor, isoproturon, metribuzin, simazine

Extraction Technique QuEChERS

Berry

Compounds Multiresidue (27-herbicides)

Olive oil

Matrix

Separation Technique

LC: Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μm particle size).

LC: Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm, 5 μm particle size)

HPLC: Purosphere RP-18 (55 mm × 2 mm × 3 μm).

HPLC: C8 (100 × 2.1 mm I.D., 1.8 μm particle size) and C8 (150 × 4.6 mm, 5 μm particle size)

Detection Technique

TOF-MS

TOF-MS

API-Q-Trap-MS: positive ESI mode

IT-Q-MS

Figures of Merit and Remarks

Linearity: range 5–500 μg/kg (r2 ranged 0.988–0.999). Recoveries: n.a. RSDs: (intraday): 0.9–4%; RSDs (inter-day): 3.5–9%. LODs: 0.4–20 μg/kg (except: bromoxynil 20 μg/kg, fluroxypyr 45 μg/kg, trifluralin 85 μg/kg).

Linearity: range LOQs: 500 μg/kg (r2 ranged 0.997–0.999). LODs: 0.01–5 μg/kg. LOQs: 0.03–10 μg/L. CV: 6.5–36.3%. Matrix effect study. Recoveries good (except for less polar <70%). Calibration range 0.1– 50.0 ng/L (r2 ranged 0.9888–0.9997). Mean recoveries (2.5–50 μg/kg): atrazine (81.0–89.7%); butachlor (61.3–67.7); Isoproturon (83.4–90.8); metribuzin (36.3–58.7); simazine (78.1–89.8). Precision: HorRat (25 μg/kg): 0.06–0.24. Uncertainty components (expressed as relative measures, calculated at 25 ng/g): 0.08–0.14. Exact mass library database; LODs: 5–100 μg/kg.

(continued)

[156]

[155]

[154]

[153]

References

148 Diquat, paraquat, chormequat

2,4-D, 2,4-DB, 2,4-DP, clopyralid, dicamba, MCPP, MCPA, picloram, triclopyr

Olive oil

Vegetation

Onion

SLE-SPE

n-Hexane/10 mM HFBA aq soln partitioning

MAE with a domestic microwave. SPE: Florisil.

ISTISAN 23/97 guidelines

MMLLE-MIP

Atrazine, propazine and simazine

Alachlor, ametryn, atrazine, chlorpropham, chlorthaldimethyl, fluazifop-butyl, isopropalin, metolachlor, metribuzin, molinat, oxadiazon, propazine, propham, propyzamide, simazine, terbuthylazine, terbutryn Diquat, paraquat, chormequat

Extraction Technique QuEChERs

Compounds Multiresidue (76-herbicides)

Red grapefruit

Matrix Apple, green pepper, red, pepper, kiwi, lettuce, pear, tomato, strawberry, aubergine, banana, tangerine, cucumber, marrow, persimmon Apple and lettuce

LC: Kromacil 100–5C18 (150 × 2.1 mm I.D.)

IP-HPLC: Xterra C8 column (100 × 21 mm, 3 μm)

GC: DB-5MS (30 m × 0.25 mm I.D., df film thickness 0.25 μm)

MS2

MS: EI Ion source (70 eV). Full scan (35–500 m/z) SIM mode. MS-ESI mode

qMS

[161]

Linearity: r > 0.996. Recovery: >92% (RSD > 7%). RSDs: repeatability <5% reproducibility <9%.LOSs: 4 μg/kg. Chlormequat 0.3 μg/kg Average recoveries: 53–80%

[162]

[160]

[159]

[158]

References [157]

Recoveries: 69.2–105.0%, RSDs: <10.7%. LOQ: 0.003–0.015 mg/kg

Extraction efficiency: 79% (simazine); 98% (atrazine); 86% (propazine). RSDs: 0.72–1.55% LODs: 22–38 μg/kg. —

UV-Vis (λ = 220 nm)

HPLC: C18 column (4.6 mm × 25 cm, 5 μm)

GCxGC

Figures of Merit and Remarks Accurate-mass databases for screening

Detection Technique TOF-MS

Separation Technique HPLC: XDB-C18 column (4.6 mm × 50 mm, 1.8 μm particle size)

Table 5.1. Summary of sample preparation, detection techniques, and figures of merit for herbicides analysis. (cont.)

149

Garlic

Buturon, chlorbromuron, chlortoluron, diuron, fenuron, fluometuron, isoproturon, linuron, metobromuron, metoxuron, monolinuron, monuron, neburon, siduron I, siduron II, tebuthiuron Atrazine, prometryn, simazine

1-(3–4-Dichlorophenyl)-3methylurea, 1-(3–4-dichlorophenyl)-urea, 1-(3-chloro-4-methylphenyl)3-ethylurea, 1-(3-chloro-4methylphenyl)-urea, 1-(4-isopropylphenyl)-3methylurea, 1-(4-isopropylphenyl)-urea, atrazine, chlortoluron, desethylatrazine, desethylterbuthylazine, desisopropylatrazine, diuron, isoproturon, linuron, simazine, terbuthylazine

Rice and corn

Infant nutrient cereal-based foods

Oysters

Forages, smooth bromegrass and switchgrass

Compounds 2,4-D-butylate, acetochlor, alachlor, atrazine, butachlor, chlorotoluron, molinate metolachlor, nitrofen, pendimethalin, pretilachlor, prometryn, oxadiazon, oxyfluorfen, simazine, trifluralin Isoxaflutole (balance) and its metabolites

Matrix

Extraction Technique

PLE

PMAE vs. UE, AMAE, SE

SLE-SPE cleanup: Florisil SPE columns (6 mL, 1000 mg)

LLE-SPE

MAE with a domestic microwave. SPE: Florisil.

HPLC: Zorbax Eclipse XDB-C8 (150 mm × 4.6 mm I.D., 5 μm). LC: octadecyl bonded silica Uptispher ODB stationary phase (50 mm × 2 mm, 3 μm) GC

HPLC: Zorbax Eclipse XDB-C18 column (5 μm particle size, 250 mm × 4.6 mm I.D.)

MS-MS MS

MS: ESI-MS: MRM mode

MS-MS: heated nebulizer APCI interface operated in the negative ion mode. MRM mode. Posthotochemical derivatization. Fluorescence detector: λex = 350 nm; λem 450 nm.

HPLC: Luna C8(2) column (30 mm × 2 mm) with a 3 μm diameter particle size.

Detection Technique MS: EI Ion source (70 eV). Full scan (35–500 m/z) SIM mode.

Separation Technique GC: DB-5MS (30 m × 0.25 mm I.D., df film thickness 0.25 μm)

Figures of Merit and Remarks

[165]

Linearity: r > 0.9980. Recoveries: 75.3–104.3% (rice), 75.0–105.1% (corn). RSD: 1.5–9.6% (rice), 0.9–9.9% (corn). Intraday precision: 1.5–7.1%, Inter-day precision: 6.4–15.6%. Better results with PMAE than with AMAE, UE and SE Recoveries: 66.2–88.6% RSD: ≤12.62%. RSD: 14–66% Recovery: 32–46% for phenylureas and 29–75% for triazines.

[167]

[166]

[164]

[163]

References

Recoveries: 94–105%, LOQs: 0.3 μg/kg. LODs: 0.01– 0.05 μg/kg

Linearity: 0.01–5.0 mg/L (r2 > 0.995). Recoveries: 69.0–105.4%, RSDs: 1.0–10.4%. LODs: 1–3 μg/ kg

150 Carbendazim and cyprodinil

HFSLME chlorhydric acid-methanol (80:20, v/v)

SLE zinc acetate dihydrate DLLME chlorobenzene and acetone

Captan, folpet, and captafol

Cucumber, tomato, and pepper

SUSME decanoic acid and tetrabutylammonium decanoate

Carbendazim, thiabendazole, and fuberidazole

Oranges, limes, lettuces, lemons, apples, bananas, grapefruits, pears, potatoes, and tangerines Apples

PLE acetone–n-hexane (25:75, v/v)

MAE chlorhydric acid SPE charcoal activated minicolumn (I.D. = 0.8 cm)

Benomyl and carbendazim

Isoprothiolane

PLE ethyl acetate

Extraction Technique

Isoprothiolane

Compounds

Polished rice

Spinach, komatsuna, qing geng cai, broccoli, cauliflower, green pepper, and okra Orange flesh extract and tap water

Matrix

LC

GC

LC

GC

GC

Separation Technique

ESI-MS

ECD

Fluorescence λ excitation: 286 and 350 nm λ emission: 320 and 350 nm

ECD

Fluorimetry λ emission: 418 and 308 nm

MS (SIM)

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis.

[20]

LOD: 17.4 ng mL−1 Recoveries: 91–106% RSD: ≤2% LOD: 0.012 ppm LOQ: 0.04 ppm Recoveries: 106–126% RSD: <7% LOQ: 14.0, 1.3, and 0.03 μg kg−1 Recoveries: 93–102% RSD: 2.8–3.5%

LOD: 3–8 μg kg−1 Recoveries: 93–110% RSD: 3.8–4.9% LOD: 0.1–0.3 μg kg−1 LOQ: 0.4–1.0 μg kg−1 RSD: 3.4–19%

[16]

LOD: 3 μg kg−1 Recoveries: 79–99% RSD: 10–18%

[25]

[24]

[23]

[21]

References

Figures of Merit and Remarks

151

Pyrimethanil, cyprodinil, and mepanipyrim Carbendazim, thiabendazole, imazalil, tridemorph, triadimefon, bitertanol, prochloraz, flutriafol, myclobutanil, iprodione, diphenylamine, and procymidone Thiabendazole

Chlorothalonil, vinclozolin, hetalaxyl, penconazole, procymidone, myclobutanil, propiconazole, tebuconazole, and ioprodine

Wine

Fruit-based baby food and juices

Brussels sprouts

Oranges (fruit and juice), grapes, lemon, and strawberries

Hymexazol, drazoxolon, vinclozolin, chlozolinate, oxadixyl, and famoxadone

Compounds

Wine, must, and fruit juices

Matrix

SLE acetone LLE dichlorometane

Fruits: SLE acetonitrile Juices: centrifuged

Baby food: QuEChERS Juices: SPE Oasis HLB

MIP-SPE

(1) SBSE (PDMS) (2) MASE hexane

Extraction Technique

LC (MIPS) Imprinted microspheres packed into a stainless steel LC column (50 × 4.6 mm I.D.) GCxGC 1D BPX5 (30 m × 0.25 mm, 0.25 μm) 2D BPX50 (1.0 m × 0.15 mm, 0.15 μm)

LC

LC

UPLC Zorbax Eclipse XDB-C18 (50 mm × 4.6 mm, 1.8 μm)

Separation Technique

NPD

Fluorescence λ excitation: 305 nm λ emission: 345 nm

ESI-TOF/MS

UV λ = 230–270 nm

DAD λ = 200 nm

Detection Technique

[54]

LOD: 28–74 ng L−1 LOQ: 96–246 ng L−1 Recoveries: 66–106% RSD: 2–8%

(continued)

[46]

[35]

[28]

[27]

References

LOD: 0.03 μg L−1 Recoveries: 89–102% RSD: 3–4%

LOD SBSE: 0.05–2.5 μg L−1 LOQ SBSE: 0.15–8 μg L−1 Recoveries: 83–113% RSD: 5.3–7.9% LOD MASE: 2.5–12 μg L−1 LOQ MASE: 8–40 μg L−1 RSD: 8.4–9.5% LOD: 0.026 μg mL−1 LOQ: 0.088 μg mL−1 Recoveries: 80–90% RSD: 1.6–7% LOD: 0.1–4 μg kg−1 Recoveries: 78–105% RSD: 1.7–13.8%

Figures of Merit and Remarks

152 Thiabendazole

Tetraconazole, penconazole, cyproconazole, and myclobutanil Iprodione

Chlorothalonil

Ziram

Tomato, orange, and apple juices

Apple, cucumber, and eggplant

Cucumber, tomato, apple, and peach

Cabbage, potato, and tomato

Compounds

Lemon and orange

Matrix

Digested chlorhydric acid LLE acetone

SLE methanol ELISA

SLE methanol ELISA

ELISA

SLE acetonitrile

Extraction Technique MIP-CEC Fused-silica capillary, 100 μm I.D., 375 μm O.D.

Separation Technique

SWV

UV λ = 450 nm

UV λ = 450 nm

UV λ = 490–650 nm

DAD λ = 305 nm

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.) Figures of Merit and Remarks LOD: 0.04–0.05 mg kg−1 LOQ: 0.14–0.18 mg kg−1 Recoveries: 85–105% RSD: 4.1–6.9% LOD: 0.1–0.7 μg L−1 Recoveries: 62–135% Coefficient of variation: ≤30% LOD: 0.3 ng g−1 Recoveries: 92.5–146.2% Coefficient of variation: 0.8–10.8% LOD: 0.052 ng g−1 Recoveries: 87.6–146.2% Coefficient of variation: 1.1–19.6% LOD: 0.0072 μg mL−1 Recoveries: 93–98% RSD: 3.4% Buffer: 2.0 mL of pH 2.78 Britton-Robinson Mercury drop electrode

[79]

[69]

[68]

[67]

[65]

References

153

Azoxystrobin, bitertanol, imazalil, prochloraz, and thiabendazole

Apple, grape, grapefruit, lemon, orange, pear, green pepper, persimmon, and tomato Honey

Lemon and raisin

Thiophanate-methyl

Wheat and barley grains and apples

Carbendazim, thiabendazole, oxadixyl, imazalil, and cyprodinil

Diethofencarb

Azoxystrobin

Compounds

Grapes, must, and wine

Matrix

SLE methanol/water 0.1% formic acid (80:20, v/v) SPE Oasis HLB

OCLLE: SLE acetone and SPE Chem Elut

Grains: SLE acetonitrile Fruits: SLE acetonitrile, SPE silica gel Complex formation: triethylamine and cobalt(II) acetate (1)Peels: direct DESI-MS (2)Extracts: QuEChERS

Grapes: SLE methanol and SPE C18 Wines: SPE C18

Extraction Technique

UPLC Acquity BEH C18 (2.1 mm × 100 mm, 1.7 μm)

LC

Separation Technique

MS/MS

ESI-MS/MS

IT-MS and MS/MS

Decision Limit: 0.579 ng g−1 Detection capability: 0.751 ng g−1 Recoveries: 80% RSD: 18% LOQ: 0.01 mg kg−1 Recoveries: 70–107% RSD: 3–16%

LOD: 0.5–6.5 μg kg−1 RSD: <15%

LOD: Grape: 6 μg kg−1 Wine: 2.4 μg L−1 Must: 2.4 μg L−1 LOQ: Grape: 0.021 μg kg−1 Must: 18 μg L-1 Wine: 8 μg L−1 Recoveries: 84–111% RSD: 1.5–2.6% Flow injection-solid phase spectroscopy LOD: 0.5 μg mL−1 Recoveries: 86.6–98.4% RSD: ≤1%

Optosensor λ excitation: 374 nm λ emission: 467 nm

UV-Vis λ = 360 nm

Figures of Merit and Remarks

Detection Technique

(continued)

[107]

[102]

[95]

[88]

[85]

References

154

Grape, eggplant, cucumber, apple, carrot, lemon, nectarine, potato, strawberry, green salad, spinach, tomato, and pepper

Cucumber, peppers, and tomato SLE ethyl acetate (pH 6–7)

SLE dichlorometane

MSPD C18-bonded silica

QuEChERS

Thiabendazole, propiconazole, prochloraz, oxadixyl, metalaxy, iprodione, imazalil, and carbendazim Thiabendazole, imazalil, thiophanate-methyl, pyrimethanil, procymidone, triadimefon, myclobutanil, triadimenol, fenarimol, dichlofluanid, epoxiconazole, iprodione, kresoxim-methyl, prochloraz, and diniconazole Metalaxyl, vinclozoline, tetraconazole, penconazole, procymidone, oxadixyl, propiconazole, tebuconazole, and iprodione Carbendazim, cyprodinil, diethofencarb, fludioxonil, imazalil, metalaxyl, myclobutanil, oxadixyl, prochloraz, propamocarb, pyrimethanil, tebuconazole, and thiabendazole

Apple, banana, grape, lettuce, orange, and tomato

SPE Bond Elut LRC-C18

Folpet, chlorothalonil, and quinomethionat

Apple and cherry juices and peach nectar Green peppers, tomatoes, and oranges

SPE Carbograph 4TM

Extraction Technique

Carbendazim, propiconazole, and pentachlorophenol

Compounds

Bovine whole milk

Matrix

LC

LP-GC CP-Sil 8 CB low-bleed (10 m × 0.53 mm, 0.25 μm)

LC

LC

LC

LC

Separation Technique

MS/MS

EI-MS/MS

QIT-ESI-MS

MS/MS

DAD λ = 220 and 260 nm

ESI-MS/MS

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

[169]

LOD: 0.1–0.5 μg kg−1 LOQ: 0.3–1.7 μg kg−1 Recoveries: 72.5–127% RSD: 4.7–16.4% LOQ: 0.01 mg kg−1 Recoveries: 87–105% RSD: <28%

[170]

[168]

[151]

[126]

[123]

References

LOD: 1–30 μg kg−1 LOQ: 4–100 μg kg−1 Recoveries: 71–102% RSD: <13%

Figures of Merit and Remarks LOD & LOQ: 0.03–0.1 μg L−1 Recoveries: 71–104% RSD: 4–10% LOD: 0.5–1 μg kg−1 Recoveries: 93.8–99.5% RSD: <3.4% LOD: 0.5–12 μg kg−1 RSD: 6%

155

Benomyl and mancozeb

Kresoxim-methyl, tetraconazole, tolylfluanid, tebuconazole, cyprodinil, captan, difenoconazole, thiram, penconazole, and trifloxystrobin Azoxystrobin, kresoxim-methyl, and trifloxystrobin

Shrub and nonshrub cucumbers

Apples for baby-food production

Grapes and wine

Azoxystrobin, kresoxim-methyl, trifloxystrobin, pyraclostrobin, famoxadone, and fenamidone

Vinclozolin, metalaxyl, thiabendazol, captan, procymidone, imazalil, oxadixyl, tebuconazole, and iprodione Pyrimethanil, tetraconazole, cyprodinil, penconazole, captan, myclobutanil, fludioxonil, propiconazole, tebuconazole, pyrazophos, bitertanol, and prochloraz Hymexazol, drazoxolon, vinclozolin, chlozolinate, oxadixyl, and famoxadone

Compounds

Grapes and wines

Beer, malt, and whisky

Apples

Onion and tomatoes

Matrix

SLE/LLE acetonitrile-water (90:10, v/v) LLE ethyl ether and chlorhydric acid SPE silica

(1) GC: SLE ethyl acetate (2) LC: SLE acetonitrile HP-GPC

SLE n-hexane and n-butyl acetate

SLE/LLE ethyl acetatehexane (50:50, v/v)

DI-SPME (CW-DVB and PDMS)

Modified QuEChERS

SLE ethyl acetate SPE NH2 adsorbent

Extraction Technique

LC

GC-MS LC-MS/MS

GC

LC

GC

GC CP-Sil 8 CB (15 m × 0.15 mm, 0.15 μm)

LP-GC HP-5 (10 m × 0.32 mm, 0.25 μm)

Separation Technique

DAD λ = 207 nm

MS MS/MS

ECD

DAD λ = 207 nm

MS (SIM)

MS

MS/MS (SIM)

Detection Technique

(continued)

[177]

[176]

[175]

[174]

[173]

[172]

[171]

LOD: 0.1–0.4 μg kg−1 Recoveries: 102–109% RSD: 1.9–3.7% LOD: 0.32–5.51 ng mL−1 LOQ: 1.07– 18.35 ng mL−1 RSD: <20% LOD: 0.006 to 0.3 μg L−1 Recoveries: 86–116% RSD: 1.4–11% LOD: 0.1–0.2 mg kg−1 LOQ: 0.3–0.8 mg kg−1 Recoveries: 89–105% RSD: <10% Study of fungicide residues in different regions of Iran LOD: 0.001– 0.004 mg kg−1 Recoveries: 87–110% RSD: 4–17% LOD: 0.073– 0.150 mg kg−1 Recovery: 53–164% RSD: 1.2–2.6%

References

Figures of Merit and Remarks

156 Carbendazim

Vinclozolin

Bitertanol, bupirimate, captan, chlorothalonil, cyprodinil, dichlofluanid, fenarimol, iprodione, metalaxyl, penconazole, procymidone, tebuconazole, tetraconazole, and thiabendazole Azoxystrobin, cymoxanil, fenhexamid, flusilazole, folpet, metalaxyl, ofurace, oxadixyl, procymidone, pyrimethanyl, tebuconazol, trifloxystrobin, and vinclozolin Imazalil, thiabendazole, carbendazim, prochloraz, thiophanate-methyl, tebuconazole, difenoconazole, metalaxyl, myclobutanil, iprodione, fenhexamid, dimethomorph, tolylfluanid, penconazole, dichlofluanid, and tolclophos-methyl

Carrots

Wines

Apples, six brands of fruit purees, and various types of fruit baby food

Orange, mandarin, lime, lemon, grapefruit, pomelo, and kumquat

Grapes

Vinclozoline, metalaxyl, penconazole, procymidone, fludioxinil, oxadixyl, propiconazole, tebuconazole, iprodione, and hexaconazole

Compounds

Avocado

Matrix

(1) SLE ethyl acetate (pH 6–7) (2) SLE acetonitrile, LLE hexane

SLE methanol SPE Superclean C8

SLE ethyl acetate HP-GPC

SPE PS-2 (poly-(methacrylate)-based) HPLC column RSpak DE-613 SPE PS-2

SLE acetone–petroleum ether-dichloromethane (1:1:1, v/v/v) SPE amino modified C18

(1) SLE ethyl acetate– cyclohexane mixture (1:1, v/v) (2) PLE ethyl acetate– cyclohexane (1:1, v/v) GPC

Extraction Technique

(1) LC (2) GC

LC

DAD λ = 220 nm

MEKC Fused-silica capillary, 58.3 cm (50.0 cm effective length), 75 μm I.D., 375 μm O.D. Cyclodextrin-modified MEKC Fused-silica bubble cell capillary, 64.5 cm (56 cm effective length), 75 μm I.D. GC

(1) MS/MS (2) ECD/NPD

DAD λ = 207 nm

ECD-NPD

UV λ = 230 nm

EI/CI-MS/MS

Detection Technique

LP-GC CP-Sil 8 CB low-bleed (10 m × 0.53 mm, 0.25 μm)

Separation Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

[182]

[183]

1- LOQ: 0.01 mg kg−1 2- LOD: 0.01–0.1 mg kg−1

[181]

[180]

[179]

[178]

References

LOD: 0.389–4.667 μg L−1 Recoveries: 20.2–121.7% RSD: <7%

LOQ: 0.003–0.01 m kg−1 Recoveries: 70–110%

LOD: <0.3 mg L−1 RSD: <1.3%

Figures of Merit and Remarks LOD: 0.01–2.50 μg kg−1 LOQ: 0.04–8.33 μg kg−1 Recoveries: 70–110% RSD: <19% LOD: 35–43 μg L−1 Online concentration: SW, SRW and SRMM.

157

Maneb and ethylenethiourea (ETU)

Azoxystrobin

Procymidone, pyrifenox, pyrimethanil, and thiabendazole Azoxystrobin, picoxystrobin, dimoxystrobin, kresoximmethyl, pyraclostrobin, and trifloxystrobin

Apples, grape, and tomato (fruit juices)

Grapes

Nectarine and peach

Wheat

Grape, lettuce, tomato, and strawberry

Bupirimate, cyhalothrin-lambda, dichlofluanid, dicloran, fenarimol, imazalil, metalaxyl, myclobutanil, ofurace, oxadixyl, penconazole, procymidone, propiconazole, pyrazophos, pyrimethanil, and tebuconazole Fludioxonil, bitertanol, flutriafol, cyproconazole, myclobutanil, and tebuconazole

Compounds

Fruit-based baby foods (mixed tropical fruit)

Matrix

(1) SLE ethyl acetate (2) SPE C18

(1) SPE silica (2) SLE/LLE 10 mL acetonitrile-dichlormethane (1:1, v/v) SPE C18 SLE acetone LLE dichloromethane SPE column: anhydrous sodium sulfate, silica gel, filtration aid, and active charcoal (2:1.5:1) SLE water-acetone (50:50, v/v) SPE C18

(1) SPE C18 (2) SBSE (PDMS)

SLE ethyl acetate

Extraction Technique

ESI-MS/MS (SIM)

CE Fused-silica capillary, 75 cm, 75 μm I.D., 375 μm O.D.

DART-TOF-MS DESI-LIT-MS

ECD

GC

DAD λ = 232 nm (ETU) λ = 280 nm (maneb)

DAD λ = 214 nm

MEKC Fused-silica capillary, 57 cm (50 cm effective length), 75 μm I.D.

LC

TOF-MS

Detection Technique

LV-DMI-GC J&W DB5-MS (20 m × 0.18 mm, 0.18 μm) or (30 m × 0.25 mm, 0.25 μm)

Separation Technique

[189]

LOQ: 5–30 μg kg−1 Recoveries: 78–92% RSD: 8–15%

(continued)

[188]

[187]

[186]

[185]

[184]

References

LOD: 0.005–0.2 μg mL−1 Recoveries: 58–99% RSD: 9–18%

LOQ SPE: 0.3–0.5 μg mL−1 Recoveries SPE: 31–66% RSD SPE: 8–19% LOQ SBSE: 1 μg mL−1 Recoveries SBSE: 12–35% RSD SBSE: 3–17% LOD: 0.1 and 0.01 mg L−1 Recoveries: 90–101% RSD: 0.7–3.8% LOD: 3 ng mL−1 LOQ: 10 ng mL−1 Recoveries: 86–103% RSD: 2–7%

Lowest calibration level: 0.0125 μg mL−1 Recoveries: 79–114% RSD: <20%

Figures of Merit and Remarks

158

Grapes and orange juices

Korean ginseng

Pencycuron, dazomet, tolclofosmethyl, metalaxyl, cyprodinil, tolyfluanid, procymidone, thifluzamide, flutolanil, kresoxim-methyl, fenhexamid, trifloxystrobin, difenoconazole, and azoxystrobin Pyrimethanil, cyprodinil, and pyrifenox

Thiabendazole and carbendazime

Mancozeb, maneb, and zineb

Cabbage, lettuce, pears, persimmons, spinach, and strawberries Cucumbers

DI-SPME (PDMS-DVB)

SLE acetonitrile SPE activated LC-Florisil

SLE acetonitrile, dichlorometane, and petroleum ether

SLE acetone Photocatalysis with titanium dioxide (black light 180 min) QuEChERS Derivatized with dimethyl sulphate

(1) SLE ethyl acetate– cyclohexane mixture (1:1, v/v) (2) PLE ethyl acetate– cyclohexane (1:1, v/v)

Vinclozoline, metalaxyl, tetraconazole, penconazole, procymidone, hexaconazole, bupirimate, krexosim-methyl, oxadixyl, propiconazole, tebuconazole, and iprodione Iprodione

SLE acetone DI-SPME (CW-TPR)

Extraction Technique

o-Phenylphenol

Compounds

Dry basil

Apple, grapes, oranges, and tomatoes Custard apple, mango, and kiwi

Matrix

CE Fused-silica capillary, 87 cm, 50 μm I.D.

GC

MEKC Fused-silica capillary, 57 cm (50 cm effective length), 75 μm I.D., 375 μm O.D.

LC

LC

CE Fused-silica capillary, 90 cm, 75 μm I.D. LP-GC CP-Sil 8 CB low-bleed (10 m × 0.53 mm, 0.25 μm)

Separation Technique

ESI-MS

ECD NPD

DAD λ = 228 nm

ESI-MS/MS

UV-Vis λ = 230 nm

EI/CI-MS/MS

ESI-MS (SIM)

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

LOD: 0.09–0.12 μg mL−1 Recoveries: 45–66% RSD: <11%

LOD: 0.24 ng g−1 LOQ: 0.80 ng g−1 Recoveries: 71–101% RSD: 1.8–9.8% LOD: 0.2 and 0.7 μg mL−1 LOQ: 0.7 and 2.4 μg mL−1 Recoveries: 91.7–110.2% RSD: 0.7–11.2% LOD: 0.001–0.03 ppm LOQ: 0.003–0.1 ppm Recoveries: 72.3–117.2% RSD: 0.2–3.9%

Figures of Merit and Remarks LOQ: 0.05–5 mg kg−1 Recoveries: 8–97% RSD: 3–16% LOD: 0.01–3.75 μg kg−1 LOQ: 0.03–6.17 μg kg−1 Recoveries: 70–110% RSD: <18% Recovery: 103%

[196]

[195]

[194]

[193]

[192]

[191]

[190]

References

159

Captan Pyrimethanil

Ethylenethiourea and isopropylenethiourea degradation products of the ethylenebisdithiocarbamate (EBDC) and propylenebisdithiocarbamate (PBDC)

Apples

Persimmon

Potatoes; fruit-based, cereal-based, and meat-based infant foods; potato chips; tinned potatoes; pizza; and yogurt Brewed green tea

Tomatoes

Guazatine

Oranges

Tebuconazole, bitertanol, propiconazole, pyriproxyfen, and mycrobutanil Captan and procymidone

Pyrimethanil, metalaxyl, cyprodinil, penconazole, procymidone, fludioxonil, tebuconazole, and iprodione

Compounds

Lettuces, Swiss chards, and spinaches

Matrix

SLE acetone LLE petroleum ether and dichloromethane

SBSE (PDMS)

SLE acetone and hexane LLE hexane GPC SLE methanol-water (8:2, v/v) LLE dichloromethane SPE Florisil SLE dichloromethane -LLE isooctane

LLE acetone-water 1% formic acid

SLE acetonitrile SPE Superclean Envi-Carb II/ PSA

Extraction Technique

LTM-GC DB-5 (10 m × 0.18 mm, 0.18 μm) GC

LC

LC

GC

LC

PTV-GC

Separation Technique

ECD

EI-MS

APCI-MS

UV-Vis λ = 268 nm

MS

ESI-MS/MS

IT-MS

Detection Technique

LOD: <10 ng L−1 Recoveries: 18–42% RSD: 7.6–11% LOD: 0.01 ng mL−1 Recoveries: 83 and 86% RSD: 5.6 and 1.4%

LOD: 0.02 ppm LOQ: 0.07 ppm Recoveries: 87–92% RSD: 4.5–12% LOQ: 0.01 mg kg−1 Recoveries: 90–108% RSD: 3.2–13.1%

LOD: <0.001– 0.003 mg kg−1 LOQ: 0.001– 0.007 mg kg−1 Recoveries: 79–100% RSD: 1–10% LOD: 0.50 μg kg−1 LOQ: 0.65 μg kg−1 Recoveries: 81–104% RSD: 2–8% MDL: 0.026 pg g−1 Recovery: 81%

Figures of Merit and Remarks

(continued)

[203]

[202]

[201]

[200]

[199]

[198]

[197]

References

160 Fenarimol, fludioxonil, hexaconazole, imazalil, iprodione, metalaxyl, myclobutanil, penconazole, procymidone, pyrazophos, tebuconazole, tolclofos-methyl, vinclozoline, captan, and cyprodinil Cyprodinil, fludioxonil, and tebuconazole Malachite green

Kresoxim-methyl, tebuconazole, dithianon, trifloxystrobin, pyrimethanil, dodine, cyprodinil, captan, difenoconazole, thiram, tolylfluanid, and mancozeb Captan, chlorothalonil, dichlofluanid, imazalil, iprodione, procymidone, tolclofos-methyl, thiabendazole, vinclozolin, and tolylfluanid

Cabbage and radish

Fish (carp, salmon, and trout)

Apples

Oranges, apples, peaches, pears, grapefruits, lettuce, tomato, cabbage, potato, onion, and leek

Peppers

Chlorothalonil and its degradation product 4-hydroxy-2,5,6trichloroisonaphthonitrile (4OH-TPN)

Compounds

Cucumber and aqueous environmental samples

Matrix

SLE acetone and cyclohexane-ethyl acetate GPC

(1) LC: SLE acetonitrile (2) GC: SLE ethyl acetate, HP-GPC

SLE acetone and ethyl acetate-cyclohexane (1:1, v/v) SLE ethanol Oxidation on column by PbO2

Water: SPE Presep-C Agri Cucumber: SLE watermethanol (20:80, v/v) containing 0.1% formic acid, SPE Presep-C Agri Modified QuEChERS

Extraction Technique

GC

LC-MS/MS GC-MS

GC

GC

LC

Separation Technique

MSD

MS/MS MS (SIM)

CCD-DAD λ = 400–700 nm

NPD

MS (SIM)

APPI-MS

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

Study of pesticide residues in food in Croatian markets

Study of dissipation rates under cold storage conditions Calibration by means of algorithms based on residual bilinearization (RBL) and unfolded partial least-squares/ residual bilinearization (U-PLS/RBL) LOQ: 0.004–0.01 mg kg−1 Recoveries: 83–110% RSD: 4–17%

Figures of Merit and Remarks MDL: 0.18 and 3.2 ng g−1 Recoveries: 89–105% RSD: 3.8–7.5% LOQ: 0.002–0.03 mg kg−1 Recoveries: 80–107% RSD: <15%

[209]

[208]

[207]

[206]

[205]

[204]

References

161

Azoxystrobin

Fenhexamid, pyrimethanil, tolylfluanid, and kresoxim-methyl

Grapes and raisins

Field-grown strawberries

SLE methanol

SLE toluene and propan-2-ol SPE celite and activated charcoal (1:3, w/w)

Myclobutanil, propiconazole, and nuarimol

Spiroxamine

Liquid samples: LLE n-hexane- dichloromethane (1:1, v/v) Solid samples: SLE n-hexane and dichloromethane SPE Florisil SLE/LLE cyclohexanedichloromethane (9:1, v/v)

Fludioxonil and imazalil

Oranges, lemons, grapefruits, and mandarins Brewer wort, beer, malt, and spent grain

Grapes, must, and wines

SLE acetone-hexane (1:1, v/v)

Dichlofluanid and tebuconazole

Lettuce

SLE dimethylsulfoxide SPE naphthalene-(1,2′ pyridylazo)-2-naphtholnaphtalene column SLE hexane

Extraction Technique

Ziram and zineb

Compounds

Grain and potato

Matrix

LC

GC

GC

GC

GC

GC

Separation Technique

MS (SIM)

ECD

IT-MS

ECD

NPD

MS

UV-Vis λ = 550 nm

Detection Technique

LOQ: Grapes: 0.02 mg kg−1 Wine: 0.012 mg kg−1 Recoveries: 85–104% RSD: <12% LOQ: 0.02 and 0.1 mg kg−1 Recoveries: 68–130% RSD: <16% LOD: 0.001– 0.008 mg kg−1 Recoveries: 68–95% RSD: 1–20%

LOD: 2, 2.5, and 0.25 pg Recoveries: 81.2–107.4% RSD: 3.8–6.3%

LOD: 0.05 and 0.125 pm Recoveries: 96–99% RSD: 0.8 and 0.7% LOD: 0.8 and 0.3 mg kg−1 LOQ: 1.5 and 0.5 mg kg−1 Recoveries: 29 and 98% RSD: <10% Recoveries: 84–102% RSD: 6–7%

Figures of Merit and Remarks

(continued)

[216]

[215]

[214]

[213]

[212]

[211]

[210]

References

162 Pyrimethanil and imazalil

o-Phenylphenol

Guazatine mixture (20) (octamethylenediamine, iminodi(octamethylene) diamine, octamethylenebis(iminooctamethylene) diamine, and carbamonitrile) Carbendazim, thiabendazole, imazalil, prochloraz, iprodione, and two transformation products (imazalil and prochloraz metabolites)

Oranges and grapefruits

Oranges, grapefruits, and lemons

Maize and hard wheat

Fruit juices

Malachite green (MG) and leucomalachite green (LMG, metabolite)

Compounds

Channel catfish, rainbow trout, tilapia, basa, Atlantic salmon, and tiger shrimp

Matrix

SPE Oasis HLB

SLE sodium hydroxide-methanol LLE dicloromethane

SLE dichloromethane Derivatized with acid chloride SPE alumina minicolumn

SLE ammonium acetate buffer, hydroxylamine hydrochloride and p-toluenesulfonic acid LLE dichloromethane Oxidation with 2,3-dichloro-5,6-dicyano1,4-benzoquinone SPE alumina and propylsulfonic acid SLE acetone-hexane (1:1, v/v)

Extraction Technique

LC

LC

GC

GC

LC (UV) Alltima C18 (150 mm × 4.6 mm, 3 μm) (MS) YMC phenyl 3–4-5 cartridge (50 mm × 4.0 mm, 3 μm, 120 Å)

Separation Technique

TOF-MS

ESI-MS

AED in the iron selective mode EI-MS (SIM)

NPD

DAD λ = 618 nm APCI-MS

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

LOD: 0.08–0.45 μg L−1 Recoveries: 71–109% RSD: <11%

Study of the influence of fungicide concentration and treatment temperature on residue levels LOD: 3 ng g−1 (AED) 35.5 ng g−1 (MS) Recoveries: 101–106% RSD: 3–8% LOQ: 0.002– 0.010 mg kg−1 Recoveries: 78–86% RSD: 0.8–6.3%

Figures of Merit and Remarks LOD: 1 ng g−1 (UV) 0.25 ng g−1 (MS) Recoveries: 86–94% RSD: ≤11%

[221]

[220]

[219]

[218]

[217]

References

163

Bitertanol, carbendazim, cyprodinil, fenpropimorph, fludioxinil, hexaconazole, imazalil, metalaxyl, myclobutanil, oxadixyl, prochloraz, propamocarb, propiconazole, pyrimethanil, tebuconazole, and thiabendazole Iprovalicarb

Wines

Fluquinconazole and pyrimethanil

Thiabendazole

Triadimenol, penconazole, propiconazole, hexaconazole, cyproconazole, myclobutanil, fenarimol, and bitertanol

Mango, apple, and papaya

Orange juice and rind

Tomato puree and lemon juice

Cabbage

Fenamidone, pyraclostrobin, and trifloxystrobin

Compounds

Grapes, must, and wines

Matrix

Juice: SLE alumina, ethyl acetate Rind: MSPD alumina SPE Bond Elut LRC SLE/LLE acetone and ethyl acetate–cyclohexane (50:50, v/v)

SLE ethyl acetate–n-hexane (1:1, v/v)

SLE acetone LLE hexane SPE neutral alumina

LLE acetonitrile SPE PSA and graphite carbon black

SLE/LLE ethyl acetatehexane (50:50, v/v)

Extraction Technique

LC

LC

LC

LC

UPLC ACQUITY UPLC BEH C18 (100 × 2.1 mm, 1.7 μm)

LC

Separation Technique

ESI-MS/MS

Fluorescence λ excitation: 305 nm λ emission: 345 nm

UV-Vis λ = 254 nm

UV-Vis λ = 215 nm

ESI-MS/MS

MS (SIM)

Detection Technique

LOQ: 0.1 μg g−1 Recoveries: 82–86.5% SD: 0.3–0.15 LOD: 0.03–0.05 mg kg−1 LOQ: 0.05–0.10 mg kg−1 Recoveries: 80–95% RSD: <20% LOD: 0.15 μg kg−1 LOQ: 0.50 μg kg−1 Recoveries: 87–97% RSD: 4.8–7.4% LOQ: Tomato: 0.005 mg kg−1 Lemon: 0.01 mg kg−1 RSD: 5.2–10.8%

LOQ: Grapes: 0.02–0.03 mg kg−1 Must: 0.02 mg L−1 Wine: 0.04–0.06 mg L−1 Recoveries: 80–113% RSD: <11.3% LOD: 0.3–3.3 μg L−1 LOQ: 0.7–6.7 μg L−1 Recoveries: 63–109% RSD: 1–13%

Figures of Merit and Remarks

(continued)

[227]

[226]

[225]

[224]

[223]

[222]

References

164 Propiconazole, fenbuconazole, cyproconazole, difenoconazole, tebuconazole, hexaconazole, bromuconazole (both stereoisomers), epoxiconazole, tetraconazole, triticonazole triadimefon, triadimenol, and myclobutanil

Spiroxamine

Captan and folpet

Ferbam, maneb, and zineb

Grapes, must, and wine

Khaki (flesh and peel) and cauliflower

Grain and red beans

Compounds

Apples, peaches, flour, raw water, and tap water

Matrix

SLE dimethyl sulfoxide Complexation with trans-1,2-diaminocyclohexane-N,N,N′,N′ tetraaceticacidmonohydrate

SLE acetone SPE Supelclean EnviChrom P

(1) SLE/LLE cyclohexane– dichloromethane (9:1, v/v) (pH 8.5–9.5) (2) SLE acetone, dichloromethane and petroleum ether

Apple/flour: SLE methanol– water (1:1, v/v) Peaches: SLE acetonitrile SPE Superclean LC-18, Oasis MCX and Oasis MAX Water: cleanup only with MCX and MAX SPE

Extraction Technique

NCI-MS

DAD λ = 196 nm

CE Fused-silica capillary 57 cm, (length to the detector 50 cm), 75 μm I.D., 375 μm O.D.

IT-MS

ESI-MS/MS

Detection Technique

GC

GC

LC

Separation Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.) Figures of Merit and Remarks LOD: Apples: 0.6–6 ppb Peaches: 0.6–8 ppb Flour: 0.2–9 ppb Water: 0.003–0.10 ppb LOQ: Apples: 2–22 ppb Peaches: 2–28 ppb Flour: 0.7–32 ppb Water: 0.01–0.30 ppb Recoveries: 57–121% RSD: ≤23% LOD(1): 0.005 mg kg−1 LOQ(1): 0.02 mg kg−1 LOD(2) grapes: 0.03 mg kg−1 LOQ(2) grapes: 0.10 mg kg−1 Recoveries: 78–102% RSD: <6% LOD: 0.01 mg kg−1 LOQ: 0.05 mg kg−1 Recoveries: 82–106% RSD: <11% LOD: 0.0013, 0.0022, and 0.0023 mM RSD: <3%

[231]

[230]

[229]

[228]

References

165

Flumorph (two isomers)

o-Phenylphenol, diphenyl, thiabendazole, imazalil, and its major metabolite R14821 (IMZ-M) Benomyl

Azoxystrobin, trifloxystrobin, kresoxim-methyl, tebuconazole, tetraconazole, flusilazole, fenhexamid, and dimetomorph

Tridemorph, carbendazim, thiabendazole, imazalil, propiconazole, and bitertanol

Lemon, orange, and grapefruit

Grapes

Apple puree, concentrated lemon juice, and tomato puree

Banana and orange

Compounds

Cucumber, tomato, and natural water

Matrix

SLE acetone

SLE/LLE acetone and ethyl acetate–cyclohexane (50:50, v/v) (pH 6)

(1) SLE acetone, dichloromethane and petroleum ether, SPE LC-NH2 (2) SLE acetone:water (1:1, v/v), SPE C18

SLE diethyl ether (pH 12)

Vegetables: QuEChERS Water: SPE C18

Extraction Technique

LC

LC

Single wavelength LC

LC

LC

Separation Technique

ESI-MS/MS

ESI-MS/MS

UV-Vis λ = 235 nm

APPI-MS

UV-Vis λ = 242 nm

Detection Technique

[232]

LOD: 0.004 mg kg−1 (isomer) LOQ: 0.01 mg kg−1 (isomer) 0.02 mg kg−1 (flumorph) Recoveries: 98–101% Coefficient of variation: 1.6–6.2% LOD: 0.01 μg g-1 and 0.05 μg g−1 (diphenil) Recoveries: 67–100% RSD: 2–8% LOD: 54 μg kg−1 LOQ: 163 μg kg−1 Recoveries: 23–143% RSD: ≤23% SPE columns lab made Lowest calibration levels: juices and purees: 1–10 μg kg−1 concentrated lemon juice: 2–20 μg kg−1 Recoveries: 77–106% RSD: <15% LOD: 0.005– 0.025 mg kg−1 LOQ: 0.05 mg kg−1 Recoveries: 75–99% RSD: <13%

(continued)

[236]

[235]

[234]

[233]

References

Figures of Merit and Remarks

166 Cyproconazole, diniconazole, tetraconazole, thiabendazole, flusilazole, triadimenol, triadimefon, carbendazim, and the degradation product 2-aminobenzimidazole Imazalil, prochloraz, and degradation products Penconazole, myclobutanil, cyproconazole, triadimefon, triadimenol, imazalil, tebuconazole, hexaconazole, flusilazole, epoxiconazole, bitertanol, propiconazole, and prochloraz Malachite green (MG) and leucomalachite green (LMG)

Chlorothalonil, vinclozolin, furalaxyl, and oxadixyl

Wine (rose, red, and white)

Lettuce

Carp muscles

Orange and lemon Wine

Malachite green (MG) and leucomalachite green (LMG)

Compounds

Salmon muscles

Matrix

SLE ethyl acetate

LV-DMI-PTV-GC DB5-MS (20 m × 0.18 mm, 0.18 μm)

LC

LC

Centrifugation

SLE hydroxylamine hydrochloride, p toluenesulfonic acid and acetate buffer LLE dichloromethane SPE Strata SCX

LC

LC

LC

Separation Technique

QuEChERS

SLE McIlvaine buffer (pH 3), 1-pentanesulfonic acid, N, N,N′,N′-tetramethyl-1,4phenylenediamine dihydrochloride and acetonitrile SPE aromatic sulfonic acid-bonded SPE Oasis HLB

Extraction Technique

TOF-MS

UV-Vis (MG) λ = 620 nm Fluorescence (LMG) λ excitation: 265 nm λ emission: 360 nm

TOF-MS IT-MS/MS MS-MS

MS

APPI-MS

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

LOD: 0.25–5 ng mL−1 LOQ: 0.25–7.5 ng mL−1 Recoveries: 80–120% RSD: <14% Decision limit: 0.15 and 0.13 μg kg−1 Detection capability: 0.37 and 0.32 μg kg−1 Recoveries: 62–90% RSD: <11% Lowest calibration level: 0.005 mg kg−1 Recoveries: 73–118% RSD: <10%

Study of metabolites

LOD: 2–8 μg L−1 LOQ: 9–31 μg L−1 Recoveries: 83–109% RSD: <10%

Figures of Merit and Remarks LOD: 0.15 μg kg−1 Recoveries: 70–85% RSD: 3.1–1.3% Use of an oxidative pre-column reactor

[242]

[241]

[240]

[239]

[238]

[237]

References

167

Captan, thiabendazole, and procymidone Cyprodinyl, penconazole, captan, kresoxim-methyl, myclobutanil, tebuconazole, and bitertanol Pyrimethanil, cyprodynil, penconazole, kresoxim-methyl, and myclobutanil Multiresidues (18). See Excel file for further information (Website address located at the end of the chapter). Mycrobutanil, propiconazole, tebuconazole, and bitertanol

Pyrimethanil, vinclozolin, metalaxyl, procymidone, bupirimate, oxadixyl, and iprodione

Captan, pricymidone, and thiabendazole

Apples

Apples

Apple-based baby food

Brewed green tea

Celeriac and carrot

Apple and peach

Compounds

Peach

Matrix

SLE ethyl acetate HP-GPC

SLE ethyl acetate

SBSE (PDMS)

(1) Buffered QuEChERS (2) SLE ethyl acetate, GPC

(1) SLE acetonitrile, SPE Bond-Elut NH2 (2) QuEChERS

SLE Acetonitrile SPE Bond-Elut NH2

SLE ethyl acetate HP-GPC

Extraction Technique GC DB5-MS (20 m × 0.18 mm, 0.18 μm) PTV-GC Rg: 1 m × 0.32 mm I.D. CP-Sil 8 CB (15 m × 0.15 mm, 0.15 μm) PTV-GC Rg: 1 m × 0.32 mm I.D. CP-Sil 8 CB (15 m × 0.15 mm, 0.15 μm) PTV-LP-GC Rc: 3 m × 0. 25 mm I.D. Rtx-5 Sil MS (10 m × 0.53 mm, 0.5 μm) Dual LTM-GC DB-5 (10 m × 0.18 mm, 0.18 μm) DB-17 (10 m × 0.18 mm, 0.18 μm) GCxGC 1D: CP-Sil 5 CB (15 m × 0.25 mm, 0.25 μm) 2D: BPX50 (0.8 m × 0.1 mm, 0.1 μm) GCxGC 1D: DB-XLB (30 m × 0.25 mm, 0.25 μm) 2D: DB-17 (1 m × 0.1 mm, 0.1 μm)

Separation Technique

TOF-MS

TOF-MS

MS PFPD

HR-TOF-MS

EI-SIM

EI-SIM

HR-TOF-MS

Detection Technique

[249]

LOD: 0.7–7 μg kg−1 RSD: <6.6%

(continued)

[248]

[247]

[246]

LOD: 3–23 pg RSD: <11%

LOD: 0.0025–0.1 mg kg−1 Recoveries: 72–109% RSD: ≤26% LOD: 21–510 ng L−1 RSD: <4.85%

[245]

[244]

[243]

LOQ: 2.5–10 μg kg−1 RSD: 4.7–7% LOQ: 0.11–19 μg kg−1 Recoveries: 46–120% RSD: <26% LOD: <0.005 mg kg−1 RSD: ≤17%

References

Figures of Merit and Remarks

168 Pyrifenox, cyprodinil, and pyrimethanil

Famoxadona

Cyprodinil, imazalil, oxadixyl, thiabendazole, and carbendazim Thiophanate, metil, and carbendazim Trifloxystrobin, fenhexamid, and famoxadone

Grapes and wines

Tomato, lemon, raisin, and avocado

Cabbage and tomatoes

Tomato, grape, and wine

Compounds

Apple and orange juice

Matrix

MAE acetone LLE hexane and dichloromethane SLME/LLME cyclohexanedichloromethane (9:1, v/v)

SLE methanol-water 0.1% formic acid (80:20, v/v) SPE OASIS HLB

SLE/LLE ethyl acetatehexane (50:50, v/v)

DI-SPME (PDMS-DVB)

Extraction Technique

GC

LC

LC

GC DB-17MS (15 m × 0.25 mm, 0.15 μm)

CE Fused-silica capillary, 57 cm (length to the detector 50 cm), 50 μm I.D.

Separation Technique

NPD ECD

UV λ = 270 nm

ESI-MS/MS

MS ECD

UV λ = 214 nm

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

LOD: 0.01–0.08 mg L−11 (ECD) 0.02–0.16 mg L−1 (NPD) LOQ: 0.004–0.05 mg kg−1 (ECD) 0.01–0.1 mg kg−1 (NPD) Recoveries: 81–102% RSD: <12%

Figures of Merit and Remarks LOD: 3.1–47 μg L−1 Recoveries: 5–46% RSD: 2–6% Online preconcentration: NSM, FESI and SWMR LOD: 0.06 (ECD) and 0.02 (MS) mg L−1 Recoveries: 103% (ECD) and 96% (MS) RSD: 11.7% (ECD) and 6.8% (MS) Expanded uncertainty: <35% LOQ: 0.01 mg kg−1 Recoveries: 66–106% RSD: <20% MDL: 0.05 μg g−1 Recoveries: 68–99%

[254]

[253]

[252]

[251]

[250]

References

169

Fludioxonil, procymidine, and carbendazim

Malachite green, gentian violet, and their leuco-metabolites

Pyrimethanil, procymidone, nuarimol, fenarimol, benalaxyl, and penconazole

Pyrimethanil, cyprodinil, and tebuconazole

Chlorothalonil, fenarimol, iprodione, metalaxyl, myclobutanil, penconazole, procymidone, propiconazole, and tebuconazole

Grass carp, eel, salmon, shrimp, and shellfish

Tomato

Apples

Grapes

Compounds

Grapes and lettuces

Matrix

SLE ethyl acetate SPE PSA

Modified QuEChERS

SLE McIlvaine buffer (pH 3), p-toluenesulfonic acid, N,N,N′,N′-tetramethyl-1,4phenylenedi-amine dihydrochloride, acetonitrile LLE dichloromethane SPE OASIS MCS SLE acetone DI-SPME (PDMS-DVB)

SLE water-acetone (50:50, v/v) SPE C18

Extraction Technique

GCxGC 1D: nonpolar RTX-5MS (10 m × 0.18 mm, 0.2 μm) 2D: polar TR-50MS (1 m × 0.1 mm, 0.1 μm)

GC CP-Sil 8 CB (15 m × 0.15 mm, 0.15 μm)

MEKC Fused-silica capillary 57 cm (50 cm detection length), 50 μm I.D.

LC

RM-MEKC Fused-silica capillary 60 cm (50 cm effective length), 75 μm I.D., 375 μm O.D.

Separation Technique

TOF-MS

MS

DAD λ = 210–240 nm

ESI-MS/MS

DAD λ = 230 nm

Detection Technique

LOD: 0.134– 0.373 mg kg−1 Recoveries: 94–102% RSD: 1.8–5.3% Online preconcentration: REPSM LOD: 0.03–0.98 ng mL−1 Recoveries: 94–102% RSD: 2–8% Use of protectants, protect coinjected analytes against degradation, adsorption, or both. LOD: 0.7–3 ng g−1 Recoveries: 78–103% RSD: 4–8%

LOD: 0.002– 0.01 μg mL−1 Recoveries: 70–92% RSD: 0.22–10.01% Online preconcentration: SW, SRW, SRMM Decision limits: 0.02–0.10 μg kg−1 Detection capability: 0.04–0.13 μg kg−1 Recoveries: 81–116% RSD: 2–18%

Figures of Merit and Remarks

(continued)

[259]

[258]

[257]

[256]

[255]

References

170 Malachite green and leucomalachite green

SLE acetonitrile, ascorbic acid, and perchloric acid LLE dichloromethane SPE Strata-x 33 μm polymeric column

Grapes: SLE ethyl acetatehexane (1:1, v/v), SPE Supelclean Envi-Carb-II/ PSA Wines: SPE Supelclean Envi-Carb-II/PSA

Benalaxyl, benalaxyl-M, boscalid, cyazofamid, famoxadone, fenamidone, fluquinconazole, iprovalicarb, pyraclostrobin, trifloxystrobin, and zoxamide

Edible goldfish muscle

SLE water DI-SPME (PDMS-DVB)

Azoxystrobin, metominostrobin, kresoxim-methyl, trifloxystrobin, picoxystrobin, dimoxystrobin, and pyraclostrobin

Baby food: carrots, vegetables, chicken with rice, chicken with vegetables, and lamb with vegetables Grapes and wines

SLE acetonitrile SPE Supelclean Envi Carb-II/ PSA

Extraction Technique

Cyprodinil, fludioxonil, iprodione, metalaxyl, penconazole, pyrimethanil, procymidone tebuconazole, triadimefon, triadimenol, and vinclozolin

Compounds

Lettuce, Swiss chard and spinach

Matrix

LC

GC

GC

GC

Separation Technique

ESI-IT-MS (MRM)

IT-MS

MS

IT-MS

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

LOD: <0.001– 0.036 mg L−1-kg−1 LOQ: 0.001–0.98 mg L−1 kg−1 Recoveries: 59–129% RSD: <16% Use of protectants LOD: 0.13 and 0.06 ng g−1 Recoveries: 72–113% RSD: 5.4–10.8%

Figures of Merit and Remarks LOD: <0.001– 0.005 mg kg−1 LOQ: 0.001–0.01 mg kg−1 Recoveries: 89–133% RSD: <10% LOD: 1–30 pg mL−1 LOQ: 0.01–0.4 ng g−1 Recoveries: 96–109% RSD: 2.3–8.4%

[263]

[262]

[261]

[260]

References

171

Malachite green and leucomalachite green

Carbendazim

Tebuconazole, difenoconazole, and azoxystrobin

Chlorothalonyl, metalaxyl, penconazole, procymidone, myclobutanil, iprodione, propiconazole, tebuconazole, and vinclozolin Hexaconazole

Wheat grain

Green tea

Spinach

Black tea

Compounds

Rainbow trout

Matrix

LLE n-hexane-acetone (1:1, v/v) SPE Florisil

SLE acetone LLE dichlorometane

(1) SLE methanol-chlorhydric acid, LLE dichlorometane (2) SLE methanol-chlorhydric acid, SPE Diol Absorbex (3) MSPD Silica gel PLE acetone-n-hexane (20:80, v/v)

SLE acetonitrile-methylene chloride ASPEC alumina and propylsulfonic acid

Extraction Technique

GCxGC 1D: BPX5 (18 m × 0.25 mm, 0.25 μm) 2D: BPX50 (0.75 m × 0.15 mm, 0.15 μm) GC

GC

LC

LC

Separation Technique

NPD

NPD ECD

ECD

DAD λ = 279 nm

ESI-MS/MS

Detection Technique

Instrumental LOD: 0.05 mg kg−1 LOQ: 0.1 mg kg−1 Recoveries: 81–96% RSD: 0.3–5%

LOD: 0.008–0.141 ppm LOQ: 0.030–0.563 ppm Recoveries: 94–110% RSD: <0.2–4% RSD: 1–9%

Decision limit: 0.13 and 0.16 μg kg−1 Detection capability: 0.22 and 0.27 μg kg−1 Recoveries: 58–110% RSD: 4.8–7.3% LOQ: 0.02 μg g−1 Recoveries: 71.2–90.7% RSD: 3.1–5.2%

Figures of Merit and Remarks

(continued)

[268]

[267]

[266]

[265]

[264]

References

172 Thiabendazole

Tetraconazole and diniconazole

Triflumizole

Metalaxyl-M, penconazole, folpet, diniconazole, propiconazole, difenoconazole, and azoxystrobin Triforine

Dimethyldithiocarbamates (DMDs), ethylenebis(dithiocarbamates) (EBDs), and propylenebis(dithiocarbamates) (PBDs)

Tomatoes and green beans

Apple, pear, and cucumber

Wines

Apple, tomato, and tinned blackcurrants

White grapes, cucumbers, tomatoes, and rucola

Compounds

Orange

Matrix

SLE sodium hydrogen carbonate and DLpenicillamine (pH 12)

SLE ethyl acetate

SPE OASIS HLB DLLME trichloroethane and acetone

SLE methanol LLE dichloromethane SPE Florisil

SLE methanol LLE methylene chloride SPE Hyflo Super Cel LLE methylene chloride

SLE ethyl acetate

Extraction Technique

LC

LC

GC

LC

GC

Separation Technique

ESI-MS

ESI-MS/MS

μ-ECD MS

UV λ = 238 nm

ECD

EEM fluorescence λ excitation: 272–304 nm λ emission: 324–384 nm

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.) Figures of Merit and Remarks LOD: 0.89–2.0 ng mL−1 Recoveries: 99–103% Two algorithms for calibration: alternating trilinear decomposition (ATLD) and the alternating normalizationweighted error Limit of determination: 0.001 ppm Recoveries: 83–100% LOD: 0.02 mg kg−1 Recoveries: 87.5–93% RSD: <6% LOQ: 20–120 ng L−1 (MS) LOQ: 40–250 ng L−1 (ECD) Recoveries: 61–99% RSD: 1–15% LOD: 0.005 mg kg−1 Recoveries: 57–100% RSD: 4.1–17% LOD: 0.03 mg kg−1 LOQ: 0.05 mg kg−1 Recoveries: 64–106% RSD: <11%

[274]

[273]

[272]

[271]

[270]

[269]

References

173

Carbendazim and thiabendazole

Carbendazim and benomyl

Dimethomorph

Lemons

Champinion, rye, and wheat

Dried hops

SLE acetone and methanolchlorhydric acid or acetone-hydrochloric LLE dichloromethane or dichloromethane-n-hexane LC Zorbax Rx C8 SLE acetone LLE dichloromethane LLP hexane GPC SPE Florisil SPE Isolute aminopropyl

SLE ethyl acetate LLE chloroform

SLE acetone-water (5:1, v/v) DI-SPME (CW-TPR)

SLE acetone and ethyl acetate-hexane (1:1, v/v)

Famoxadone, fluquinconazole, and trifloxystrobin Dichloran, flutriafol, o phenylphenol, prochloraz, and tolclofos methyl

SLE sodium hydrogen carbonate and DLpenicillamine (pH 12)

Extraction Technique

Dimethyldithiocarbamates (DMDs), ethylenebis(dithiocarbamates) (EBDs), and propylenebis(dithiocarbamates) (PBDs)

Compounds

Cherries, lemons, oranges, and peaches

Apples, pears, grapes, cherry tomatoes, cocktail tomatoes, cucumbers, tomatoes, tamarillos, papaya, and broccoli Red wine

Matrix

MSD (SIM)

DAD λ = 279 nm

LC

GC

DAD λ = 210 nm

DAD λ = 210 nm APCI-MS APCI-IT-MS/MS

ECD DAD λ = 230 nm

ESI-MS/MS

Detection Technique

CE Fused-silica capillary 64.5 cm (56 cm effective length), 50 μm I.D.

LC

GC LC

LC

Separation Technique

LOD: 0.045 ppm LOQ: 0.1 ppm Recoveries: 79–103% RSD: <10%

(continued)

[280]

[279]

[278]

[277]

[276]

[275]

LOD: 0.5–2 μg kg−1 LOQ: 2–7.9 μg kg−1 Recoveries: 97–101% RSD: 0.1–-5.4%

LOQ: 0.05–0.1 μg mL−1 Recoveries: 73–79% RSD: 0.5–13% LOQ: 0.25–1 mg kg−1 (DAD) LOQ: 0.0005– 0.01 mg kg−1 (MS-MS/MS) Recoveries: 5–69% RSD: <15% LOD: 2.0 and 2.3 μg mL−1 LOQ: 2.5 μg mL−1 Recoveries: 67 and 62% RSD: <9% LOQ: 0.02–0.1 μg g−1 Recoveries: 75–85% RSD: 2.5–3.7%

References

Figures of Merit and Remarks

174 Dichloran, flutriafol, o phenylphenol, prochloraz, and tolclofos methyl

Oranges, lemons, bananas, peppers, chards, and onions

White wines

Cyprodinil, dichlofluanid, folpet, fludioxonil, metalaxyl, penconazole, pyrimethanil, procymidone, tebuconazole, and vinclozolin Cyprodinil and fludioxonil

White grapes

MSPD C8

DI-SPME (DVB–CAR–PDMS)

SLE dichloromethane-acetone (75:25, v/v)

SLE water

Procymidone

Fosetyl-aluminum (Al)

SLE/LLE methylene chloride

Dichloran, flutriafol, o phenylphenol, prochloraz, and tolclofos-methyl

Chards, onions, peppers, bananas, lemons, and oranges Agricultural formulations and wine

Lettuces

MSPD C–8

Imazalil (α + β)

Oranges

SLE ethyl acetate-hexane (50:50, v/v) SLE acetonitrile SPE Sep-Pak Plus PS-2

Extraction Technique

Thiabendazole

Compounds

Oranges

Matrix

LC

GC

GC

LC

LC

CE Fused-silica capillary 64.5 cm (effective length 56 cm), 75 μm I.D.

GC

Separation Technique

APCI-MS (SIM) UV λ = 210 nm

MS (SIM)

MS (SIM)

ESI-MS/MS

DPP

MS

DAD λ = 200 nm

NPD

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

LOD: 2.4 × 10−9 M Recoveries: 94–100% SD: 0.011–0.021 LOD: 0.05 mg kg−1 LOQ: 0.2 mg kg−1 Recoveries: 98–106% RSD: <10% LOD: 1–18 μg kg−1 LOQ: 2–38 μg kg−1 Recoveries: 75–116% RSD: <10% LOD: 0.1 and 0.9 μg L−1 LOQ: 0.2 and 2 μg L −1 Recoveries: 100% RSD: 5% LOQ: 0.01–0.1 mg kg−1 Recoveries: 40–99% RSD: <12%

Figures of Merit and Remarks Recoveries: 87–106% LOD: 0.1 mg mL−1 Recoveries: 96–97% RSD: <21% Chiral resolution of Imazalil with 2-hydroxypropylβ-cyclodextrin LOQ: 0.005–0.08 mg kg−1 Recoveries: 53–77% RSD: <26%

[288]

[287]

[286]

[285]

[284]

[283]

[282]

[281]

References

175

Imazalil

Apples

Wines

Carbendazim, cymoxanil, thiophanate-methyl, metalaxyl, pyrimethanil, fludioxonil, fenhexamid, azoxystrobin, folpet, procymidone, penconazol, cyprodinil, vinclozolin, and dichlofluanid Nuarimol, triadimenol, triadimefon, folpet, vinclozolin, and penconazole

White grapes

SLE methanol

DI-SPME (PDMS–DVB)

SLE dichloromethane– acetone (75:25, v/v) SPE Sep-Pak silica

HS-SPME (PA)

Pyrimethanil and kresoxim-methyl

CITP-CZE preseparation capillary (fluorinated ethylenepropylene copolymer, FEP), 90 mm, 0.8 mm I.D. FEP analytical capillary 320 mm (240 mm detector length), 0.3 mm I.D.

LC

LC

GC

GC

MSPD C18 and silica

Captan, carboxin, fludioxonil, flutolanil, folpet, pyrimethanil, quintozene, and tebuconazole

Orange, apple, tomato, artichoke, carrot, and courgette Grapes, strawberries, tomatoes, and ketchup

Separation Technique CE Fused-silica capillary, 150 cm, 75 μm I.D.

Extraction Technique SLE methanol-water SPE C8

Procymidone and thiabendazole

Compounds

Apples, grapes, oranges, pears, strawberries, and tomatoes

Matrix

UV λ = 220 nm

DAD λ = 200–225 nm

DAD λ = 200–380 nm

MS (SIM)

ECD NPD

ESI-MS (SIM)

Detection Technique

LOD: 4–27 μg L−1 Recoveries: 95–103% RSD: 2.4–25.7% LOD: 0.02 μg g−1 Recoveries: 91% RSD: 2.7%

LOD: 0.1 and 0.01 μg mL−1 LOQ: 0.005–0.05 mg kg−1 Recoveries: 61–80% RSD: <4% LOD: 3–30 μg kg−1 LOQ: 10–100 μg kg−1 Recoveries: 62–102% RSD: <16% LOD: 1.8–2.0 and 2.8–3.1 ng g−1 LOQ: 5.5–6.1 and 8.5–9.4 ng g−1 Recoveries: 90–107% RSD: 7.4–15% LOD: 0.01–0.31 mg kg−1 LOQ: 0.02–0.64 mg kg−1 Recoveries: 41–88% RSD: ≤28%

Figures of Merit and Remarks

(continued)

[294]

[293]

[292]

[291]

[290]

[289]

References

176 Chlorothalonil, metalaxyl, penconazole, procymidone, iprodione, oxadixyl, and propiconazole Maneb, zineb, mancozeb, thiram, and ziram

Wines

Triadimefon, propiconazole, myclobutanil, and penconazole Propamocarb

Carbendazim, dichlofluanid, diethofencarb, imazalil, kresoxim-methyl, myclobutanil, oxamyl, penconazole, propiconazole, pyrimethanil, tebuconazole, and thiabendazole Bitertanol, carboxin, flutriafol, pyrimethanil, tebuconazole, and triadimefon

Strawberries and wine Lettuce, radish, leek, and cabbage

Grape, kiwi fruit, strawberry, spinach, lemon, peach, and nectarine

Grapes

Multiresidues (22). See Excel file for further information.

Cereals

Tobacco and peaches

Compounds

Matrix

(1) SPE C18 (2) SBSE (PDMS)

LC

LC Polymer based Shodex RSpak DE-613 column (150 × 6 mm) packed with polymethacrylate gel (6 μm) LC

SLE methanol

SLE ethyl acetate

GC

GC

GC Poraplot Q (10 m, 0.53 mm I.D.)

GC

Separation Technique

DI-SMPE (PA)

QuEChERS

MAE iso-octane , stannous chloride, and chlorhydric acid

SBSE (PDMS)

Extraction Technique

APCI-MS (SIM)

ESI-MS/MS

ESI-MS (SIM)

MS (SIM)

MS/MS

FID

MS (SIM)

Detection Technique

Table 5.2. Summary of the sample preparation, detection techniques, and figures of merit for fungicides analysis. (cont.)

LOQ SPE: 0.003–0.01 mg kg−1 LOQ SBSE: 0.01 mg kg−1 Recoveries: 15–100% RSD: ≤19%

Lowest calibration level: 0.01 mg kg−1 Recoveries: 77–84% RSD: ≤12%

LOD: 0.005–0.1 mg kg−1 Recoveries: 81–113% RSD: 0.8–20% LOQ: 0.0075 mg kg−1 Recoveries: 4–134% RSD: <20% LOD: 30–100 ng kg−1 RSD: 2–28% LOD: 25 μg kg−1 Recoveries: 88–112% RSD: <11%

Figures of Merit and Remarks LOD: <0.5 μg mL−1

[301]

[300]

[299]

[298]

[297]

[296]

[295]

References

177

Carbendazim, metalaxyl, pyrimethanil, procymidone, fenarimol, benalaxyl, penconazole, nuarimol, and azoxystrobin Fenhexamid

Vinclozolin, procymidone, myclobutanil, iprodione, and prochloraz

Chlorothalonil

Prochloraz

Fludioxonil and famoxadone

Vinclozolin and procymidone

Must and wine

Apple, strawberry, plum, orange, and lettuce

Tomatoes and cucumbers

Mango, papaya, and orange

Tomato pulp, pear puree, and concentrated lemon juice

Grapes

Compounds

Wines

Matrix

PHWE MMLLE toluene

SLE acetone Derivatized with pyridine hydrochloride LLE diethyl ether-n-hexane (1:4, v/v) LLP toluene SLE/LLE acetone and ethyl Acetate-cyclohexane (50:50, v/v)

SLE ethyl acetate

Modified QuEChERS

ELISA

DI-SPME (PDMS-DVB)

Extraction Technique

LV-GC

LC

LC GC

GC

PTV-GC CP-Sil 8 CB (15 m × 0.15 mm, 0.15 μm)

MEKC Fused-silica capillary, 57 cm (50 cm detection length), 50 μm I.D.

Separation Technique

MS

ESI-MS/MS (MRM)

UV-Vis λ = 220 nm ECD

ECD

NCI-MS

UV λ = 492 nm

DAD λ = 210 nm

Detection Technique

Lowest calibration levels: 0.002 and 0.01 mg kg−1 Recoveries: 77.1–96.5% RSD: 4.2–11.5% LOQ: 0.3–1.3 μg kg−1 Recoveries: 26–28% RSD: 10–23%

LOD: 0.054– 0.113 mg L−1 Online preconcentration: REPSM LOD: 0.13 μg L−1 Recoveries: 95–118% RSD: ≤12% LOD: 0.07– 0.619 ng mL−1 LOQ: 0.22–2.4 ng mL−1 Recoveries: 70–110% RSD: 2.8–11.3% LOD: 0.0025 mg L−1 LOQ: 0.02 mg kg−1 Recoveries: 86–114% RSD: <10.5% LOD: 0.05 mg kg−1 LOQ: 0.1 mg kg−1 Recoveries: 80–94% RSD: 5.6–12.6%

Figures of Merit and Remarks

[308]

[307]

[306]

[305]

[304]

[303]

[302]

References

178

Analysis of Endocrine Disrupting Compounds in Food

successfully used for the determination of triazine herbicide residues in potatoes [18, 63]. Ultrasonic solvent extraction (USE) [18] and pressurized liquid extraction (PLE) [63] have been used to isolate the analytes from the sample matrix. The detection limits achieved with USE followed by NACE are lower than the default value of MRL established by current European Union legislation [18]. Reversed-phase capillary electrochromatography has been applied to the determination of phenylurea herbicide residues in different samples of vegetables and vegetable processed food (i.e., soya milk and canned soya shoots) [64]. Cacho et al. [65] used the combination of molecularly imprinted and capillary electrochromatography as a powerful tool for the selective determination of the fungicide thiabendazole in citrus samples. The limits of detection (LODs) obtained with the developed method were below the established MRLs.

Detection At present, most methods for the determination of fungicide and herbicide residues in

food matrices use liquid and gas chromatography associated with a detector, mass spectrometry being the most widely used (Figure 5.1). Liquid chromatography has been commonly associated with ultraviolet (UV), fluorescence, and/or tandem mass spectrometry (MS-MS or MS2). HPLC-UV is a more universal detection system, but fluorescence is generally more selective and in some cases is more sensitive. Recently, LC-MS/MS has found more widespread application in these target residue analyses offering more sensitive detection and including additional residues (Figure 5.2). This figure shows the chromatogram of 148 pesticides, including 76 herbicides and 30 fungicides. Gas chromatography with electron capture (ECD), nitrogen-phosphorus (NPD), and mass spectrometry (MS) detection has been the most widely used technique. However, many herbicides and fungicides are thermally unstable or nonvolatile and cannot be determined by GC; therefore, there is a growing interest in applying LC-MS in control of target residues to ensure food safety.

12% 7% 42%

8%

10%

21% MS

UV-Vis

Immunoassay

ECD

Electrochemical

Others

Figure 5.1. Detectors and quantification methods used for herbicides and fungicides analysis in food (period 2004–2009). Source: SciFinder®, September 11, 2009.

Pesticides: Herbicides and Fungicides

179

Figure 5.2. Example of an LC-MS/MS run for the analysis of 148 pesticides using a hybrid triple quadrupole linear ion trap in the multiple reaction monitoring (MRM) mode. Most of the compounds are eluted in the same region of the chromatogram, showing the complexity of performing an MRM method for such a large amount of compounds in a unique LC run. Reprinted from Mezcua, M.; Malato, O.; García-Reyes, J. F.; Molina-Díaz, A.; Fernández-Alba, A. R. 2008. Accurate-mass databases for comprehensive screening of pesticide residues in food by fast liquid chromatography time-of-flight mass spectrometry. Analytical Chemistry 81(3):913–929. Used with permission.

Mass spectrometry Mass analyzers of various designs and performance are currently used for herbicide and fungicide determination [48–51], including low-resolution (single quadrupole) and highresolution (time-of-flight, or TOF) MS, as well as tandem mass (triple quadrupole, QqQ) [50,52,60] and ion trap (QIT). Preliminary cleanup procedures before determination by LC-MS are used only in very few studies due to the high selectivity of new mass spectrometers, but when needed, liquid–liquid partitioning (LLP) and solidphase extraction (SPE) are the most commonly used techniques. LLP cleanup can be efficiently applied using organic solvents such as dichloromethane and ethyl acetatecyclohexane [12].

Different determinations of herbicide and fungicide in food analysis employing mass spectrometers as detectors can be found in Tables 5.1 and 5.2.

Immunoassay During the last few years, the use of immunoassays has experienced an important increase [66]. Manclus et al. [67] have developed monoclonal immunoassays for the determination of triazole fungicides in fruit juices. The enzyme-liked immunosorbent assays (ELISAs) can determine triazole fungicides in fruit juices down to the MRLs currently legislated. Traces of iprodione residue have also been determined by ELISA in fruits and vegetables (i.e., apple, cucumber, and

180

Analysis of Endocrine Disrupting Compounds in Food

eggplant) [68]. Watanabe et al. described the analytical performance of the ELISA for chlorothalonil in fruits and vegetables. The method is reliable, cost-effective, simple, and rapid because it is possible to directly analyze target residue by diluting each sample extract with water prior to the ELISA analysis [69]. In another application of immunoassay, trace levels of chlorophenols and chloroanisoles have been determined in liquid food samples, mainly within the wine sector, by both conventional immunoassay procedures and the use of a miniaturized liquid handling system compatible with immunosensor devices. The measurement tool advantages are that it is low cost, simple to perform, and does not require highly trained personnel and expensive equipment [70]. A mass-sensitive immunosensor has been suggested for the fast and selective determination of residual amounts of chloroacetanilide herbicides in foodstuffs and environmental samples [71]. A novel immunosensor based on single-frequency impedance has been designed and developed for the determination of atrazine in wine grapes and other foodstuff products [72], and a disposable immunomembrane-based electrochemical sensor has been developed for the rapid detection of picloram in rice, lettuce, and paddy-filled water [73]. A new competitive ELISA method has been developed for the urea herbicide isoproturon in food and environmental samples [74].

the determination of trace amounts of thifensulfuron-Me in pesticide formulations, soil, and orange juice [76]. Verma et al. [77] have developed a DPP method for the determination of thiophanate-Me in both formulation and residues in foodstuffs, and Mercan and Inam [78] have developed a method for the determination of the herbicide thiazopyr in fruit juice and soil samples. A voltammetric method was developed and applied to determine the ziram content in synthetic and spiked vegetable samples [79]. Paraquat has been determined in food (i.e., apple and potato) by square wave voltammetry with a fluoroapatite-modified carbon paste electrode [80] and in natural water, food, and beverages by multiple square wave voltammetry (MSWV) [81]. A sensitive method for the determination of the herbicides nitralin and oryzalin in agricultural formulations, vegetables, and grape juice samples by adsorptive stripping voltammetry (AdSV) at a hanging mercury drop electrode has been described [82]. Differential pulse stripping voltammetry (DPSV) has been applied to determine the herbicides ametryn, cyanatryn, and dimethametryn in food and water. With the aid of chemometrics, analysis of synthetic mixtures has been successfully performed [83]. Differential pulse voltammetry has also been used for the determination of propham and maleic hydrazide herbicides in potatoes after matrix solid-phase dispersion (MSPD) [84].

Electrochemical detection An important number of works devoted to electrochemical detection and quantification have also been published. Wang et al. [75] used a polarographic catalytic wave for the determination of thiram in presence of Cu2+. The second derivative peak current of the catalytic wave set at −0.8 V (vs SCE) is linearly proportional to the thiram concentration. The suggested method is sensitive, simple, and rapid. Differential pulse polarography (DPP) has also been used for

Other detectors An opto-sensor has been reported for the fluorimetric determination of azoxystrobin residues in grapes, musts, and wines. Analyte is converted in a strongly fluorescent degradation product by UV irradiation [85]. A simple, sensitive, and rapid flame atomic absorption spectrometric (FAAS) method has been described for the indirect determination of zinc ethylenebisdithiocarbamate (zineb) and ferric dimethyldithiocarbamate (ferbam)

Pesticides: Herbicides and Fungicides

in foodstuffs (i.e., water, tomato, and wheat grain) [86]. A system for rapid dichlofluanid-carrying fungicide (i.e., euparen) determination using near-infrared (IR) spectroscopy has been developed. A dry-extraction system for IR (DESIR) technique has been used, and the technique has been applied to the determination of fungicide at parts per million-(order concentration) on tomato surfaces [87]. Verma et al. [88] have developed a new, simple, rapid, and sensitive spectrophotometric method for the determination of thiophanate-Me in foodstuffs (i.e., grains and apples). The method is based on the reaction between thiophanate-Me and Co(II) in the presence of triethylamine. The yellowish-green reaction product has maximum absorbance at 360 nm. A sensitive fourth derivative spectrophotometric procedure has been developed for ziram determination in commercial samples such as Zirax and Ziron and from wheat grains [89]. Flow injection methodology has been used for the spectrophotometric determination of 2,4-dichlorophenoxyacetic acid herbicide in fruits and food samples [90]; bromoxynil herbicide in compound formulations, flour, and meal (i.e., wheat maize) [91]; and Starane (fluroxypur) herbicide in its formulations and food samples (i.e., corn, wheat flour, and maize) [92]. A simple and sensitive chemiluminescent (CL) method for the determination of dithiocarbamate fungicides in vegetables has been developed. Dithiocarbamate fungicides react with luminol to produce an intense chemiluminescence in the presence of hexacyanoferrate(III) and hexacyanoferrate(II) in alkaline solution [93]. A sensitive CL-based microimmunosupported liquid membrane assay (μ-ISLMA) has been developed that enables cleanup, enrichment, and detection of simazine in a single miniaturized cartridge system. The influence of the sample matrix has been

181

investigated on mineral water, orange juice, and milk [94]. A useful Excel file that includes all analytes and matrices can be downloaded from http://web.ua.es/es/qace/documentos/book/ herbicides-and-fungicides.xls.

Acknowledgments The authors would like to thank the Spanish Government (project no. CTQ200806730-C02-01) and the Generalitat Valenciana (projects no. ACOMP/2009/144 and A-04/09). I.P. Román also thanks Caja Mediterráneo (CAM) for his fellowship.

List of Abbreviations AdSV AED AMAE

AOAC APCI APGD

APPI ASE ASPEC C8 C18 CCD-DAD

CD

Adsorptive stripping voltammetry Atomic emission detection Atmospheric microwave assisted extraction Association of official agricultural chemists Atmospheric pressure chemical ionization Atmospheric pressure glow discharge desorption Atmospheric pressure photoionization Accelerate solvent extraction Automated solid-phase extraction cartridge Octil stationary phase Octadecil stationary phase Charged coupled device–diode array detector Circular dichroism detector

182

CE

Analysis of Endocrine Disrupting Compounds in Food

Capillary electrophoresis CEC Capillary electrochromatography CI Chemical ionization CITP-CZE Capillary isotachophoretic on-line coupled– capillary zone electrophoresis CL Chemiluminescence CNBF 4-Chloro-3,5dinitrobenzotrifluoride CW-DVB Carbowaxdivinylbencene CW-TPR Carbowaw–template resin DAD Diode array detector DART Direct analysis in a real-time ion source DBCA Dichlorobenzoic acid DDT Dichloro-diphenyltrichloroethane DESI Desorption electrospray ionization DESIR Dry-extraction system for infrared spectroscopy DI-SPME Direct immersion solid-phase microextraction DLLME Dispersive liquid– liquid microextraction DPP Differential pulse polarography DPSV Differential pulse stripping voltammetry DPV Differential pulse voltammetry DSPE Dispersive solid-phase extraction DVB-CAR-PDMS Divinylbencenecarboxenpolydimethylsiloxane ECD Electron capture detector ED Endocrine disruptor

EDC EEM EI ELISA ESI FAAS

FAO FEP FESI FID FRAC FTD GC GCxGC GPC HF-LLLME

HFM HFSLME

HP-GPC

HPLC HPTLC HR-TOF HS-SPME

Endocrine disrupting compound Excitation-emission matrix Electron impact Enzyme-linked immunosorbent assay Electrospray ionization Flame atomic absorption spectrometry Food and Agriculture Organization Fluorinated ethylene propylene Field-enhanced sample injection Flame ionization detector Fungicide resistance action committee Flame thermoionic detection Gas chromatography Two dimensional gas chromatography Gel permeation chromatography Liquid–liquid–liquid microextraction with a hollow fiber membrane Hollow fiber membrane Hollow fiber supported liquid membrane extraction High performance gel permeation chromatography High performance liquid chromatography High performance thin layer chromatography High resolution time-of-flight detection Headspace solid-phase microextracion

Pesticides: Herbicides and Fungicides

IA I.D. IP-RPLC IR IT LC LIT-MSn LLE LLP LOD LOQ LP LTM LV-DMI µECD µ-ISLMA

MAE MASE MDL MEKC MIP MIP-CEC

MMLLE

MRL MRM MS MSD MS/MS (or MS2)

Immunoaffinity Inner diameter Ion-pair, reversed-phase liquid chromatography Infrared spectroscopy Ion trap Liquid chromatography Linear ion trap tandem mass spectrometry Liquid–liquid extraction Liquid–liquid partitioning Limit of detection Limit of quantification Low pressure Low thermal mass Large volume–difficult matrix introduction Micro electron capture detector Micro-immuno supported liquid membrane assay Microwave assisted extraction Membrane assisted solvent extraction Method detection limit Micellar electrokinetic chromatography Molecular imprinted polymer Molecular imprinted polymer–capillary electrochromatography Microporous membrane liquid– liquid extraction Maximum residue limit Multiple-reaction monitoring Mass spectrometry Mass selective detector Tandem mass spectrometry

MSPD MSWV NACE NCI NPD NSM OCLLE O.D. PA PCB PDMS PDMS-DVB

PFPD PHWE PLE PMAE PSA PTV QIT qMS QqQ QuEChERS

REPSM Rg RM-MEKC

183

Matrix solid-phase dispersion Multiple square wave voltammetry Nonaqueous capillary electrophoresis Negative chemical ionization Nitrogen-phosphorus detector Normal stacking mode On-column liquid– liquid extraction Outer diameter Polyacrylate Polychlorinated biphenyl Polydimethylsiloxane Polydimethylsiloxanedivinylbenzene Pulsed flame photometric detection Pressurized hot water extraction Pressurized liquid extraction Pressurized microwave assisted extraction Primary-secondary amine Programmable temperature vaporizer Quadrupole ion trap Single quadrupole mass spectrometry Triple quadrupole Quick, Easy, Cheap, Effective, Rugged and Safe sample preparation method Reversed electrode polarity stacking mode Retention gap Reverse migration– micellar electrokinetic chromatography

184

Analysis of Endocrine Disrupting Compounds in Food

RRLC RSD SBSE SCE SE SEC SIM SLE SLME SPE SPME SRM SRMM SRW

SUSME

SW SWMR SWV TLC TOF TSD UA-MSPD

UPLC UE US

Rapid resolution liquid chromatography Relative standard deviation Stir-bar sorptive extraction Saturated calomel electrode Soxhlet extraction Size-exclusion chromatography Selected ion monitoring Solid–liquid extraction Solid–liquid microextraction Solid-phase extraction Solid-phase microextraction Selected reaction monitoring Stacking with reverse migration of micelles Reverse migrating micelles and a water plug Supramolecular solvent-based microextraction Sweeping Stacking with matrix removal Square wave voltammetry Thin layer chromatography Time-of-flight Thermoionic specific detector Ultrasound assistedmatrix solid phase dispersion Ultra performance liquid chromatography Ultrasonic extraction Ultrasonic solvent extraction

UV-Vis WHO

Ultraviolet-visible spectrophotometry World Health Organization

References [1] http://www.ecpa.be/en/pesticides/ [2] http://ec.europa.eu/environment/ppps/pdf/ statistics_regulation.pdf [3] http://en.wikipedia.org/wiki/Fungicide [4] http://www.frac.info/frac/publication/anhang/ FRAC_CODE_LIST.pdf [5] http://en.wikipedia.org/wiki/Herbicides [6] Codex Alimentarius: http://www.codexalimentarius.net/ mrls/pestdes/jsp/pest_q-e.jsp [7] Codex Alimentarius website: ftp.fao.org/codex/ Publications/understanding/Understanding_ EN.pdf [8] Japanese Food Chemical Research Foundation: h t t p : / / w w w. m 5 . w s 0 0 1 . s q u a r e s t a r t . n e . j p / foundation/search.html [9] Informal coordination of MRLs established in Directives 76/895/EEG, 86/362/EEG, 86/363/ EEG, and 90/462/EEG (5058/VI/98): http:// ec.europa.eu/food/plant/protection/ resources/09-99-2.pdf http://ec.europa.eu/food/ plant/protection/pesticides/regulation_ ec_396_2005_en.htm, http://www.observatoirepesticides.gouv.fr/index.php?pageid=359. [10] 40CFR180: http://www.access.gpo.gov/nara/cfr/ waisidx_02/40cfr180_02.html [11] Federal Food, Drug, and Cosmetic Act, US Food and Drug Administration website: http:// www.fda.gov/opacom/laws/fdcact/fdctoc.htm]. [12] Picó, Y.; Font, G.; Ruiz, M.J.; Fernández, M. 2006. Control of pesticide residues by liquid chromatography-mass spectrometry to ensure food safety. Mass Spectrometry Reviews 25(6):917– 960. [13] http://faostat.fao.org/ [14] Fernández-Alba, A.R.; Agüera, A. 2007. Pesticides in food. Analytical and Bioanalytical Chemistry 389(6):1661. [15] Tadeo, J.L.; Sánchez-Brunete, C.; Pérez, R.A. 2004. Herbicide residues and fungicide residues. In: Handbook of Food Analysis, 2nd ed., Nollet, L., ed. New York: Marcel Dekker, pp. 1249– 1296. [16] Tanaka, T.; Hori, T.; Asada, T.; Oikawa, K.; Kawata, K. 2007. Simple one-step extraction and cleanup by pressurized liquid extraction for gas chromatographic-mass spectrometric determination of pesticides in green leafy vegetables. Journal of Chromatography A 1175(2):181–186. [17] Bester, K.; Bordin, G.; Rodríguez, A.; Schimmel, H.; Pauwels, J.; VanVyncht, G. 2001. How to overcome matrix effects in the determination of pesticides in fruit by HPLC-ESI-MS-MS. Analytical and Bioanalytical Chemistry 371(4):550–555.

Pesticides: Herbicides and Fungicides

[18] Rodriguez-Gonzalo, E.; Carabias-Martinez, R.; Cruz, E.M.; Dominguez-Alvarez, J.; HernandezMendez, J. 2009. Ultrasonic solvent extraction and nonaqueous CE for the determination of herbicide residues in potatoes. Journal of Separation Science 32(4):575–584. [19] Ramos, J.J.; Rial-Otero, R.; Ramos, L.; Capelo, J.L. 2008. Ultrasonic-assisted matrix solid-phase dispersion as an improved methodology for the determination of pesticides in fruits. Journal of Chromatography A 1212(1–2):145–149. [20] Pacioni, N.L.; Sueldo Occello, V.N.; Lazzarotto, M.; Veglia, A.V. 2008. Spectrofluorimetric determination of benzoimidazolic pesticides: Effect of p-sulfonatocalix[6]arene and cyclodextrins. Analytica Chimica Acta 624(1):133–140. [21] Cho, S.-K.; Abd El-Aty, A.M.; Park, Y.-S.; Choi, J.-H.; Khay, S.; Kang, C.-A.; Park, B.-J.; Kim, S.-J.; Shim, J.-H. 2007. A multiresidue method for the analysis of pesticide residues in polished rice (Oryza sativa L.) using accelerated solvent extraction and gas chromatography and confirmation by mass spectrometry. Biomedical Chromatography 21(6):602–609. [22] Picó, Y.; Fernández, M.; Ruiz, M.J.; Font, G. 2007. Current trends in solid-phase-based extraction techniques for the determination of pesticides in food and environment. Journal of Biochemical and Biophysical Methods 70(2):117–131. [23] Moral, A.; Sicilia, M.D.; Rubio, S. 2009. Determination of benzimidazolic fungicides in fruits and vegetables by supramolecular solventbased microextraction/liquid chromatography/ fluorescence detection. Analytica Chimica Acta 650(2):207–213. [24] Zang, X.; Wang, J.; Wang, O.; Wang, M.; Ma, J.; Xi, G.; Wang, Z. 2008. Analysis of captan, folpet, and captafol in apples by dispersive liquid–liquid microextraction combined with gas chromatography. Analytical and Bioanalytical Chemistry 392(4):749–754. [25] Romero-González, R.; Pastor-Montoro, E.; Martínez-Vidal, J.L.; Garrido-Frenich, A. 2006. Application of hollow fiber supported liquid membrane extraction to the simultaneous determination of pesticide residues in vegetables by liquid chromatography/mass spectrometry. Rapid Communications in Mass Spectrometry 20(18):2701–2708. [26] Zhu, L.; Ee, K.H.; Zhao, L.; Lee, H.K. 2002. Analysis of phenoxy herbicides in bovine milk by means of liquid-liquid-liquid microextraction with a hollow-fiber membrane. Journal of Chromatography A 963(1–2):335–343. [27] Viñas, P.; Aguinaga, N.; Campillo, N.; HernándezCórdoba, M. 2008. Comparison of stir bar sorptive extraction and membrane-assisted solvent extraction for the ultra-performance liquid chromatographic determination of oxazole fungicide residues in wines and juices. Journal of Chromatography A 1194(2):178–183. [28] Baggiani, C.; Baravalle, P.; Giraudi, G.; Tozzi, C. 2007. Molecularly imprinted solid-phase extrac-

[29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

[37]

[38]

[39]

185

tion method for the high-performance liquid chromatographic analysis of fungicide pyrimethanil in wine. Journal of Chromatography A 1141(2): 158–164. Tamayo, F.G.; Casillas, J.L.; Martin-Esteban, A. 2005. Clean up of phenylurea herbicides in plant sample extracts using molecularly imprinted polymers. Analytical and Bioanalytical Chemistry 381(6):1234–1240. Herrero-Hernández, E.; Carabias-Martínez, R.; Rodríguez-Gonzalo, E. 2009. Use of a bisphenol-A imprinted polymer as a selective sorbent for the determination of phenols and phenoxyacids in honey by liquid chromatography with diode array and tandem mass spectrometric detection. Analytica Chimica Acta 650(2):195– 201. Turiel, E.; Tadeo, J.L.; Martin-Esteban, A. 2007. Molecularly imprinted polymeric fibers for solidphase microextraction. Analytical Chemistry 79(8):3099–3104. Barker, S.A.; Long, A.R.; Short, C.R. 1989. Isolation of drug residues from tissues by solid phase dispersion. Journal of Chromatography A 475(2):353–361. Barker, S.A. 2000. Matrix solid-phase dispersion. Journal of Chromatography A 885(1–2):115– 127. Garcia-Lopez, M.; Canosa, P.; Rodriguez, I. 2008. Trends and recent applications of matrix solidphase dispersion. Analytical and Bioanalytical Chemistry 391(3): 963–974. Gilbert-López, B.; García-Reyes, J.F.; OrtegaBarrales, P.; Molina-Díaz, A.; Fernández-Alba, A.R. 2007. Analyses of pesticide residues in fruit-based baby food by liquid chromatography/ electrospray ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry 21(13):2059–2071. Ji, F.; Zhao, L.; Yan, W.; Feng, Q.; Lin, J.-M. 2008. Determination of triazine herbicides in fruits and vegetables using dispersive solid-phase extraction coupled with LC-MS. Journal of Separation Science 31(6–7):961–968. Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F.J. 2003. Fast and easy multiresidue method employing acetonitrile extraction/ partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. Journal of AOAC International 86(2): 412–431. Posyniak, A.; Zmudzki, J.; Mitrowska, K. 2005. Dispersive solid-phase extraction for the determination of sulfonamides in chicken muscle by liquid chromatography. Journal of Chromatography A 1087(1–2):259–264. Díez, C.; Traag, W.A.; Zommer, P.; Marinero, P.; Atienza, J. 2006. Comparison of an acetonitrile extraction/partitioning and “dispersive solidphase extraction” method with classical multiresidue methods for the extraction of herbicide residues in barley samples. Journal of Chromatography A 1131(1–2):11–23.

186

Analysis of Endocrine Disrupting Compounds in Food

[40] http://www.chem.agilent.com/en-US/Products/ consumables/samplepreparation/sampliqspe/ sampliqquechers/Pages/default.aspx [41] Khan, A.; Pereira, L.; Aspey, S.; Bunn, R.; Lewis, R. 2009. QuEChERS dispersive solid phase extraction for the GC/MS analysis of pesticides in grapes. Thermo Scientific Application Note: ANGSC PESTGRAPES 0709 (http:// www.thermo.com/eThermo/CMA/PDFs/Product/ productPDF_52045.pdf). [42] Koesukwiwat, U.; Sanguankaew, K.; Leepipatpiboon, N. 2008. Rapid determination of phenoxy acid residues in rice by modified QuEChERS extraction and liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta 626(1):10–20. [43] Steiniger, D.; Lu, G.; Butler, J.; Phillips, E.; Fintschenko, Y. 2009. Multi-residue pesticide analysis in green tea using a modified QuEChERS extraction and ion trap GC/MS. Separation Science 1(10):19–25. [44] Lehotay, S.J. 2007. Determination of pesticide residues in foods by acetonitrile extraction and partitioning with magnesium sulfate: Collaborative study. Journal of AOAC International 90(2): 485–520. [45] Santilio, A.; Stefanelli, P.; Dommarco, R. 2009. Fast determination of phenoxy acid herbicides in carrots and apples using liquid chromatography coupled triple quadrupole mass spectrometry. Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes 44:584–590. [46] Turiel, E.; Tadeo, J.L.; Cormack, P.A.G.; MartinEsteban, A. 2005. HPLC imprinted-stationary phase prepared by precipitation polymerization for the determination of thiabendazole in fruit. The Analyst 130(12):1601–1607. [47] Tamayo, F.G.; Martin-Esteban, A. 2005. Selective high performance liquid chromatography imprinted-stationary phases for the screening of phenylurea herbicides in vegetable samples. Journal of Chromatography A 1098(1–2): 116–122. [48] Picó, Y.; Blasco, C.; Font, G. 2004. Environmental and food applications of LC-tandem mass spectrometry in pesticide-residue analysis: An overview. Mass Spectrometry Reviews 23(1):45–85. [49] Fernández-Alba, A.R.; García-Reyes, J.F. 2008. Large-scale multi-residue methods for pesticides and their degradation products in food by advanced LC-MS. TrAC Trends in Analytical Chemistry 27(11):973–990. [50] Picó, Y.; Barceló, D. 2008. The expanding role of LC-MS in analyzing metabolites and degradation products of food contaminants. TrAC Trends in Analytical Chemistry 27(10):821–835. [51] García-Reyes, J.F.; Hernando, M.D.; MolinaDíaz, A.; Fernández-Alba, A.R. 2007. Comprehensive screening of target, non-target, and unknown pesticides in food by LC-TOF-MS. TrAC Trends in Analytical Chemistry 26(8): 828–841.

[52] Dömötörová, M.; Matisová, E. 2008. Fast gas chromatography for pesticide residues analysis. Journal of Chromatography A 1207(1–2): 1–16. [53] Mondello, L.; Casilli, A.; Tranchida, P.Q.; Lo Presti, M.; Dugo, P.; Dugo, G. 2007. Comprehensive gas chromatography coupled to mass spectrometry for the separation of pesticides in a very complex matrix. Analytical and Bioanalytical Chemistry 389(6):1755–1763. [54] Khummueng, W.; Trenerry, C.; Rose, G.; Marriott, P.J. 2006. Application of comprehensive twodimensional gas chromatography with nitrogenselective detection for the analysis of fungicide residues in vegetable samples. Journal of Chromatography A 1131(1–2):203–214. [55] Sherma, J. 2005. Thin-layer chromatography of pesticides–A review of applications for 2002– 2004. Acta Chromatographica 15:5–30. [56] Sherma, J. 2007. Review of advances in the thin layer chromatography of pesticides: 2004–2006. Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes 42(4):429–440. [57] Jamuna, M.; Naika, M,; Jeevaratnam, K.; Bawa, A.S. 2005. Detection and determination of various fungicides in fruits and vegetables using thin layer chromatography. Journal of Food Science and Technology 42(2):205–208. [58] Juan-García, A.; Font, G.; Picó, Y. 2005. Determination of organic contaminants in food by capillary electrophoresis. Journal of Separation Science 28(9–10):793–812. [59] Boyce, M.C. 2007. Determination of additives and organic contaminants in food by CE and CEC. Electrophoresis 28(22):4046–4062. [60] Font, G.; Ruiz, M.J.; Fernández, M.; Picó, Y. 2008. Application of capillary electrophoresismass spectrometry for determining organic food contaminants and residues. Electrophoresis 29(10):2059–2078. [61] Picó, Y. 2006. Analysis of fungicides in fruits and vegetables by capillary electrophoresis-mass spectrometry. Methods in Biotechnology 19:297– 309. [62] Hernández-Borges, J.; Rodríguez-Delgado, M.A.; García-Montelongo, F.J.; Cifuentes, A. 2005. Analysis of pesticides in soy milk combining solidphase extraction and capillary electrophoresis-mass spectrometry. Journal of Separation Science 28(9–10):948–956. [63] Carabias-Martinez, R.; Rodriguez-Gonzalo, E.; Miranda-Cruz, E.; Dominguez-Alvarez, J.; Hernandez-Mendez, J. 2007. Sensitive determination of herbicides in food samples by nonaqueous CE using pressurized liquid extraction. Electrophoresis 28(20):3606–3616. [64] De Rossi, A.; Desiderio, C. 2005. Application of reversed phase short end-capillary electrochromatography to herbicides residues analysis. Chromatographia 61(5–6): 271–275. [65] Cacho, C.; Schweitz, L.; Turiel, E.; Pérez-Conde, C. 2008. Molecularly imprinted capillary electro-

Pesticides: Herbicides and Fungicides

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74] [75]

[76]

[77]

chromatography for selective determination of thiabendazole in citrus samples. Journal of Chromatography A 1179(2):216–223. Schwack, W.; Hrenn, H. 2007. “Renaissance” of a fungicide. Detection and determination of chlorothalonil-protein conjugates in tomatoes. GIT Labor-Fachzeitschrift 51(2):86–88. Manclus, J.J.; Moreno, M.J.; Plana, E.; Montoya, A. 2008. Development of monoclonal immunoassays for the determination of triazole fungicides in fruit juices. Journal of Agricultural and Food Chemistry 56(19):8793–8800. Watanabe, E.; Miyake, S. 2007. Immunoassay for iprodione: Key estimation for residue analysis and method validation with chromatographic technique. Analytica Chimica Acta 583(2):370–376. Watanabe, E.; Miyake, S.; Ito, S.; Baba, K.; Eun, H.; Ishizaka, M.; Endo, S. 2006. Reliable enzyme immunoassay detection for chlorothalonil: Fundamental evaluation for residue analysis and validation with gas chromatography. Journal of Chromatography A 1129(2):273–282. Redondo, B.A.; Pérez-Villareal, B.; Setford, S.; Nicholls, C.; Bourdin, L.; Wijdenes, J.; VermotDesroches, C. 2004. Detecting trace levels of chlorophenols and chloroanisoles in wineries by immunodiagnostic techniques. In: Rapid Methods for Biological and Chemical Contaminants in Food and Feed. van Amerongen, A., Barug, D., Lauwaars, M., eds., Weimar, TX: C.H.I.P.S. Publications, pp. 339–359. Nartova, Y.V.; Eremin, S.A.; Ermolaeva, T.N. 2008. Mass-sensitive immunosensors for determining chloroacetanilide herbicides. Journal of Analytical Chemistry 63(12):1191–1198. Rodríguez, Á.; Valera, E.; Ramón-Azcón, J.; Sanchez, F.J.; Marco, M.P.; Castañer, L.M. 2008. Single frequency impedimetric immunosensor for atrazine detection. Sensors and Actuators B: Chemical 129(2):921–928. Tang, L.; Zeng, G.-M.; Shen, G.-L.; Li, Y.-P.; Zhang, Y.; Huang, D.-L. 2008. Rapid detection of picloram in agricultural field samples using a disposable immunomembrane-based electrochemical sensor. Enviromental Science & Technology 42(4):1207–1212. Li, F.; Sun, F.; Liu, X.; Cui, H. 2007. Development of anti-isoproturon polyclonal antibody. Agricultural Sciences in China 6(8):964–969. Wang, J.; Cui, Y.-M.; Zhao, W.-J.; Yang, M.-M.; Liu, W. 2008. Determination of thiram using polarographic catalytic wave in presence of Cu(II). Fenxi Huaxue 36(4):533–536. Inam, R.; Sarigui, T.; Guleman, E.Z.; Uncu, N. 2006. Polarographic determination of herbicide thifensulfuron methyl/application to agrochemical pesticides, soil, and fruit juice. International Journal of Environmental Analytical Chemistry 86(15):1135–1149. Verma, B.C.; Chauhan, C.; Thakur, L.; Sharma, D.K. 2004. Determination of thiophanate methyl in its formulation and agricultural produce by dif-

[78]

[79] [80]

[81]

[82]

[83]

[84]

[85]

[86]

[87]

[88]

[89]

187

ferential pulse polarography. Pesticide Research Journal 16(1):90–93. Mercan, H.; Inam, R. 2008. Differential pulse polarographic behaviour of thiazopyr herbicide and application to its determination in fruit juice and soil samples. International Journal of Environmental Analytical Chemistry 88(12): 879–890. Qiu, P.; Ni, Y.N. 2008. Determination of ziram in vegetable samples by square wave voltammetry. Chinese Chemical Letters 19(11):1337–1340. El Mhammedi, M.A.; Bakasse, M.; Bachirat, R.; Chtaini, A. 2008. Square wave voltammetry for analytical determination of paraquat at carbon paste electrode modified with fluoroapatite. Food Chemistry 110(4):1001–1006. Souza, D.D.; Machado, S.A.S.; Pires, R.C. 2006. Multiple square wave voltammetry for analytical determination of paraquat in natural water, food, and beverages using microelectrodes. Talanta 69(5):1200–1207. Thriveni, T.; Rajesh Kumar, J.; Sujatha, D.; Sreedhar, N.Y. 2007. Voltammetric determination of the herbicides nitralin and oryzalin in agricultural formulations, vegetables, and grape juice samples. Food Chemistry 104(3):1304–1309. Ni, Y.; Wang, L.; Serge, K. 2009. Simultaneous voltammetric determination of three herbicides in food and water samples with the aid of chemometrics. Chemical Research in Chinese Universities 25(2):151–154. Arribas, A.S.; Bermejo, E.; Chicharro, M.; Zapardiel, A. 2007. Application of matrix solidphase dispersion to the propham and maleic hydrazide determination in potatoes by differential pulse voltammetry and HPLC. Talanta 71(1):430–436. López Flores, J.; Molina Díaz, A.; Fernóndez de Córdova, M.L. 2007. Determination of azoxystrobin residues in grapes, musts and wines with a multicommuted flow-through optosensor implemented with photochemically induced fluorescence. Analytica Chimica Acta 585(1):185–191. Tuker, A.R.; Sezer, B. 2005. Indirect determination of dithiocarbamate fungicides (zineb and ferbam) in some foodstuffs by flame atomic absorption spectrometry. Turkish Journal of Pharmaceutical Sciences 2(1):35–42. Saranwong, S.; Kawano, S. 2005. Rapid determination of fungicide contaminated on tomato surfaces using the DESIR-NIR: A system for ppm-order concentration. Journal of Near Infrared Spectroscopy 13(3):169–175. Verma, B.C.; Sood, S.; Chauhan, C.; Sharma, D.K. 2004. A simple and rapid spectrophotometric method for determination of thiophanate-methyl in commercial formulations and its residues in foodstuffs. Journal of AOAC International 87(4):811–814. Sharma, V.K.; Aulakh, J.S.; Bansal, S.; Malik, A.K.; Mahajan, R.K. 2004. Fourth derivative spectrophotometric determination of fungicide ziram (zinc(II)dimethyldithiocarbamate) in com-

188

[90]

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

Analysis of Endocrine Disrupting Compounds in Food

mercial samples and wheat grains. International Journal of Environmental Analytical Chemistry 84(14–15):1105–1110. Shah, J.; Jan, M.R.; Bashir, N. 2006. Flow injection spectrophotometric determination of 2,4-D herbicide. Journal of the Chinese Chemical Society (Taipei, Taiwan) 53(4):845–850. Jan, M.R.; Shah, J.; Bashir, N. 2006. Flow injection spectrophotometric determination of bromoxynil herbicide by diazotization method. Analytical Sciences 22(1):165–167. Shah, J.; Jan, M.R.; Bashir, N. 2006. Determination of starane (fluroxypyr) herbicide using flow injection spectrophotometry. Analytical Sciences 22(1):145–146. Nakazawa, H.; Tsuda, Y.; Ito, K.; Yoshimura, Y.; Kubo, H.; Homma, H. 2004. Determination of dithiocarbamates fungicides by reversed-phase ion-pair liquid chromatograpy with chemiluminescence detection. Journal of Liquid Chromatography & Related Technologies 27(4): 705–713. Tudorache, M.; Emnqus, J. 2006. A microimmuno supported liquid membrane assay (μISLMA). Biosensors and Bioelectronics 21(8): 1513–1520. García-Reyes, J.F.; Jackson, A.U.; Molina-Díaz, A.; Cooks, R.G. 2009. Desorption electrospray ionization mass spectrometry for trace analysis of agrochemicals in food. Analytical Chemistry 81(2):820–829. Vinayaka, A.C.; Basheer, S.; Thakur, M.S. 2009. Bioconjugation of CdTe quantum dot for the detection of 2,4-dichlorophenoxyacetic acid by competitive fluoroimmunoassay based biosensor. Biosensors & Bioelectronics 24(6):1615– 1620. Ramon-Azcon, J.; Valera, E.; Rodriguez, A.; Barranco, A.; Alfaro, B.; Sanchez-Baeza, F.; Marco, M.P. 2008. An impedimetric immunosensor based on interdigitated microelectrodes (ID mu E) for the determination of atrazine residues in food samples. Biosensors & Bioelectronics 23(9):1367–1373. Kim, S.J.; Gobi, K.V.; Tanaka, H.; Shoyama, Y.; Miura, N. 2008. A simple and versatile self-assembled monolayer based surface plasmon resonance immunosensor for highly sensitive detection of 2,4-D from natural water resources. Sensors and Actuators B-Chemical 130(1):281– 289. El-Aty, A.M.A.; Lee, G.W.; Mamun, M.I.R.; Choi, J.H.; Cho, S.K.; Shin, H.C.; Shim, J.H. 2008. Development and validation of a method for the analysis of cafenstrole and its metabolite in brown rice grains and rice straw using high-performance liquid chromatography. Biomedical Chromatography 22(3):306–315. Huang, Z.; Li, Y.; Chen, B.; Yao, S. 2007. Simultaneous determination of 102 pesticide residues in Chinese teas by gas chromatography-mass spectrometry. Journal of Chromatography B 853(1–2):154–162.

[101] Berrada, H.; Font, G.; Moltó, J.C. 2004. Application of solid-phase microextraction for determining phenylurea herbicides and their homologous anilines from vegetables. Journal of Chromatography A 1042(1–2):9–14. [102] Pirard, C.; Widart, J.; Nguyen, B.K.; Deleuze, C.; Heudt, L.; Haubruge, E.; De Pauw, E.; Focant, J.F. 2007. Development and validation of a multiresidue method for pesticide determination in honey using on-column liquid-liquid extraction and liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 1152(1–2):116–123. [103] García-Reyes, J.F.; Gilbert-López, B.; MolinaDíaz, A.; Fernández-Alba, A.R. 2008. Determination of pesticide residues in fruit-based soft drinks. Analytical Chemistry 80(23):8966–8974. [104] Jecklin, M.C.; Gamez, G.; Touboul, D.; Zenobi, R. 2008. Atmospheric pressure glow discharge desorption mass spectrometry for rapid screening of pesticides in food. Rapid Communications in Mass Spectrometry 22(18):2791–2798. [105] Taylor, M.J.; Keenan, G.A.; Reid, K.B.; Fernandez, D.U. 2008. The utility of ultra-performance liquid chromatography/electrospray ionisation time-of-flight mass spectrometry for multi-residue determination of pesticides in strawberry. Rapid Communications in Mass Spectrometry 22(17):2731–2746. [106] Botitsi, H.; Econornou, A.; Tsipi, D. 2007. Development and validation of a multi-residue method for the determination of pesticides in processed fruits and vegetables using liquid chromatography-electrospray ionization tandem mass spectrometry. Analytical and Bioanalytical Chemistry 389(6):1685–1695. [107] Pozo, O.J.; Barreda, M.; Sancho, J.V.; Hernandez, F.; Lliberia, J.L.; Cortes, M.A.; Bago, B. 2007. Multiresidue pesticide analysis of fruits by ultraperformance liquid chromatography tandem mass spectrometry. Analytical and Bioanalytical Chemistry 389(6):1765–1771. [108] García-Reyes, J.F.; Hernando, M.D.; Ferrer, C.; Molina-Diaz, A.; Fernandez-Alba, A.R. 2007. Large scale pesticide multiresidue methods in food combining liquid chromatography time-offlight mass spectrometry and tandem mass spectrometry. Analytical Chemistry 79(19):7308– 7323. [109] Soler, C.; James, K.J.; Picó, Y. 2007. Capabilities of different liquid chromatography tandem mass spectrometry systems in determining pesticide residues in food: Application to estimate their daily intake. Journal of Chromatography A 1157(1–2):73–84. [110] Yulaev, M.F.; Sitdikov, R.A.; Dmitrieva, N.M.; Yazynina, E.V.; Zherdev, A.V.; Dzantiev, B.B. 2001. Development of a potentiometric immunosensor for herbicide simazine and its application for food testing. Sensors and Actuators B: Chemical 75(1–2):129–135. [111] Lentza-Rizos, C.; Balokas, A. 2001. Residue levels of chlorpropham in individual tubers and

Pesticides: Herbicides and Fungicides

[112]

[113]

[114]

[115]

[116]

[117]

[118]

[119]

[120]

[121]

composite samples of postharvest-treated potatoes. Journal of Agricultural and Food Chemistry 49(2):710–714. Pensabene, J.W.; Fiddler, W.; Donoghue, D.J. 2000. Supercritical fluid extraction of atrazine and other triazine herbicides from fortified and incurred eggs. Journal of Agricultural and Food Chemistry 48(5):1668–1672. Djozan, D.; Mahkam, M.; Ebrahimi, B. 2009. Preparation and binding study of solid-phase microextraction fiber on the basis of ametrynimprinted polymer: Application to the selective extraction of persistent triazine herbicides in tap water, rice, maize, and onion. Journal of Chromatography A 1216(12):2211–2219. Zougagh, M.; Bouabdallah, M.; Salghi, R.; Hormatallah, A.; Rios, A. 2008. Supercritical fluid extraction as an on-line clean-up technique for rapid amperometric screening and alternative liquid chromatography for confirmation of paraquat and diquat in olive oil samples. Journal of Chromatography A 1204(1):56–61. Guillén-Casla, V.; Pérez-Arribas, L.V.; LeónGonzólez, M.E.; Polo-Díez, L.M. 2008. One- and two-dimensional direct chiral liquid chromatographic determination of mixtures of diclofopacid and diclofop-methyl herbicides. Journal of Agricultural and Food Chemistry 56(7):2303– 2309. Avramides, E.J.; Gkatsos, S. 2007. A multiresidue method for the determination of insecticides and triazine herbicides in fresh and processed olives. Journal of Agricultural and Food Chemistry 55(3):561–565. Amvrazi, E.G.; Albanis, T.A. 2006. Multiresidue method for determination of 35 pesticides in virgin olive oil by using liquid-liquid extraction techniques coupled with solid-phase extraction clean up and gas chromatography with nitrogen phosphorus detection and electron capture detection. Journal of Agricultural and Food Chemistry 54(26):9642–9651. Guardia-Rubio, M.; Ruiz-Medina, A.; MolinaDíaz, A.; Ayora-Canada, M.J. 2006. Influence of harvesting method and washing on the presence of pesticide residues in olives and olive oil. Journal of Agricultural and Food Chemistry 54(22): 8538–8544. Garcia-Reyes, J.F.; Ferrer, C.; Thurman, E.M.; Fernandez-Alba, A.R.; Ferrer, I. 2006. Analysis of herbicides in olive oil by liquid chromatography time-of-flight mass spectrometry. Journal of Agricultural and Food Chemistry 54(18): 6493– 6500. Guardia-Rubio, M.; Fernández-De Córdova, M.L.; Ayora-Cañada, M.J.; Ruiz-Medina, A. 2006. Simplified pesticide multiresidue analysis in virgin olive oil by gas chromatography with thermoionic specific, electron-capture, and mass spectrometric detection. Journal of Chromatography A 1108(2):231–239. Degelmann, P.; Egger, S.; Jurling, H.; Muller, J.; Niessner, R.; Knopp, D. 2006. Determination of

[122]

[123]

[124]

[125]

[126]

[127]

[128]

[129]

[130]

[131]

[132]

189

sulfonylurea herbicides in water and food samples using Sol?Gel glass-based immunoaffinity extraction and liquid chromatography ?ultraviolet/diode array detection or liquid chromatography-tandem mass spectrometry. Journal of Agricultural and Food Chemistry 54(6):2003–2011. Wang, P.; Jiang, S.; Liu, D.; Zhang, H.; Zhou, Z. 2006. Enantiomeric resolution of chiral pesticides by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry 54(5):1577–1583. Bogialli, S.; Curini, R.; Di Corcia, A.; Lagan, A.; Stabile, A.; Sturchio, E. 2006. Development of a multiresidue method for analyzing herbicide and fungicide residues in bovine milk based on solid-phase extraction and liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 1102(1–2):1–10. Uzer, A.; Ercag, E.; Parlar, H.; Apak, R.; Filik, H. 2006. Spectrophotometric determination of 4,6-dinitro-o-cresol (DNOC) in soil and lemon juice. Analytica Chimica Acta 580(1):83–90. Rosales-Conrado, N.; León-González, M.E.; Pérez-Arribas, L.V.; Polo-Díez, L.M. 2005. Capillary liquid chromatography of chlorophenoxy acid herbicides and their esters in apple juice samples after preconcentration on a cation exchanger based on polydivinylbenzene-N-vinylpyrrolidone. Journal of Chromatography A 1076(1–2):202–206. Topuz, S.; Ízhan, G.; Alpertunga, B. 2005. Simultaneous determination of various pesticides in fruit juices by HPLC-DAD. Food Control 16(1): 87–92. Albero, B.; Sanchez-Brunete, C.; Donoso, A.; Tadeo, J.L. 2004. Determination of herbicide residues in juice by matrix solid-phase dispersion and gas chromatography-mass spectrometry. Journal of Chromatography A 1043(2):127–133. Khrolenko, M.; Dzygiel, P.; Wieczorek, P. 2002. Combination of supported liquid membrane and solid-phase extraction for sample pretreatment of triazine herbicides in juice prior to capillary electrophoresis determination. Journal of Chromatography A 975(1):219–227. Pedrero, M.; Gálvez, R.; Rodríguez, E.; Manuel de Villena, F.J.; Pingarrón, J.M. 2001. Determination of the herbicide desmetryne in organised media by adsorptive stripping voltammetry. Talanta 53(5):991–1000. Dallüge, J.; Hankemeier, T.; Vreuls, R.J.J.; Brinkman, U.A.T. 1999. On-line coupling of immunoaffinity-based solid-phase extraction and gas chromatography for the determination of s-triazines in aqueous samples. Journal of Chromatography A 830(2):377–386. Hu, Y.; Liu, R.; Zhang, Y.; Li, G. 2009. Improvement of extraction capability of magnetic molecularly imprinted polymer beads in aqueous media via dual-phase solvent system. Talanta 79(3):576–582. Basheer, C.; Lee, H.K. 2004. Hollow fiber membrane-protected solid-phase microextraction of

190

[133]

[134]

[135]

[136]

[137]

[138]

[139]

[140]

[141]

[142]

[143]

Analysis of Endocrine Disrupting Compounds in Food

triazine herbicides in bovine milk and sewage sludge samples. Journal of Chromatography A 1047(2):189–194. Balduini, L.; Matoga, M.; Cavalli, E.; Seilles, E.; Riethmuller, D.; Thomassin, M.; Guillaume, Y.C. 2003. Triazinic herbicide determination by gas chromatography-mass spectrometry in breast milk. Journal of Chromatography B 794(2): 389–395. Díaz, L.; Llorca-Pórcel, J.; Valor, I. 2008. Ultra trace determination of 31 pesticides in water samples by direct injection-rapid resolution liquid chromatography-electrospray tandem mass spectrometry. Analytica Chimica Acta 624(1):90–96. Ishimitsu, S.; Kaihara, A.; Yoshii, K.; Tsumura, Y.; Nakamura, Y.; Tonogai, Y. 2002. Simultaneous determination of Azimsulfuron, flazasulfuron, and halosulfuron-methyl in grains, seeds, vegetables, and fruits by HPLC. Journal of Health Science 48(4):335–340. West, S.D.; Hastings, M.J.; Shackelford, D.D.; Dial, G.E. 2004. Determination of Oryzalin in Water, Citrus Fruits, and Stone Fruits by Liquid Chromatography with Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry 52(19):5781–5786. Ramón-Azcón, J.; Kunikata, R.; Sanchez, F.J.; Marco, M.P.; Shiku, H.; Yasukawa, T.; Matsue, T. 2009. Detection of pesticide residues using an immunodevice based on negative dielectrophoresis. Biosensors and Bioelectronics 24(6):1592– 1597. Valera, E.; Ramón-Azcón, J.; Sanchez, F.J.; Marco, M.P.; Rodríguez, Á. 2008. Conductimetric immunosensor for atrazine detection based on antibodies labelled with gold nanoparticles. Sensors and Actuators B: Chemical 134(1): 95–103. Valera, E.; Ramón-Azcón, J.; Rodríguez, N.; Castañer, L.M.; Sánchez, F.J.; Marco, M.P. 2007. Impedimetric immunosensor for atrazine detection using interdigitated μ-electrodes (IDμE’s). Sensors and Actuators B: Chemical 125(2): 526–537. Navalón, A.; Prieto, A.; Araujo, L.; Luis Vílchez, J. 2002. Determination of oxadiazon residues by headspace solid-phase microextraction and gas chromatography-mass spectrometry. Journal of Chromatography A 946(1–2):239–245. Vitalif, M.; Guidotti, M.; Giovinazzo, R.; Cedronet, O. 1998. Determination of pesticide residues in wine by SPME and GC/MS for consumer risk assessment. Food Additives & Contaminants: Part A 15(3):280–287. Shi, C.; Gui, W.; Chen, J.; Zhu, G. 2009. Determination of oxadiargyl residues in environmental samples and rice samples. Bulletin of Environmental Contamination and Toxicology 84(2):236–239. Marchese, S.; Perret, D.; Bafile, E.; Gentili, A.; Caretti, F.; Berardino, M. 2009. Pressurized liquid extraction coupled with LC-ESI-MS-MS for the determination of herbicides chlormequat

[144]

[145]

[146]

[147]

[148]

[149]

[150]

[151]

[152]

[153]

and mepiquat in flours. Chromatographia 70 (5/6):761–767. Martins-Júnior, H.A.; Lebre, D.T.; Wang, A.Y.; Pires, M.A.F.; Bustillos, O.V. 2009. An alternative and fast method for determination of glyphosate and aspectrometryminomethylphosphonic acid (AMPA) residues in soybean using liquid chromatography coupled with tandem mass. Rapid Communications in Mass Spectrometry 23(7):1029–1034. Potter, T.L.; Carpenter, T.; Putnam, R.; Reddy, K.; Clark, J.M. 1991. Rapid method for analysis of atrazine and acetanilide herbicides in groundwater by micro liquid/liquid extraction. Journal of Agricultural and Food Chemistry 39(12): 2184–2187. Herranz, S.; Ramón-Azcón, J.; Benito-Peña, E.; Marazuela, M.D.; Marco, M.P.; MorenoBondi, M.C. 2008. Preparation of antibodies and development of a sensitive immunoassay with fluorescence detection for triazine herbicides. Analytical and Bioanalytical Chemistry 391: 1801–1812. Sun, Y.; Luo, L.; Wang, F.; Li, J.;Cao, Y. 2009. Ion-pairing high-performance liquid chromatography determination of amitrole in apple after solid-phase extraction and precolumn derivatization. Analytical and Bioanalytical Chemistry 395(2):465–471. Barker, S.A.; Long, A.R.; Short, C.R. 1989. Isolation of drug residues from tissues by solid phase dispersion. Journal of Chromatography A 475(2):353–361. Kmellár, B.; Fodor, P.; Pareja, L.; Ferrer, C.; Martínez-Uroz, M.A.; Valverde, A.; FernandezAlba, A.R. 2008. Validation and uncertainty study of a comprehensive list of 160 pesticide residues in multi-class vegetables by liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 1215(1–2):37–50. Lehotay, S.J.; de Kok, A.; Hiemstra, M.; Van Bodegraven, P. 2005. Validation of a fast and easy method for the determination of residues from 229 pesticides in fruits and vegetables using gas and liquid chromatography and mass spectrometric detection. Journal of Aoac International 88(2): 595–614. Ferrer, I.; Rhurman, E.M.; Zweigenbaum, J.A. 2007. Screening and confirmation of 100 pesticides in food samples by liquid chromatography/ tandem mass spectrometry. Rapid Communications in Mass Spectrometry 21(23):3869–3882. Takatori, S.; Okihashi, M.; Okamoto, Y.; Kitagawa, Y.; Kakimoto, S.; Murata, H.; Sumimoto, T.; Tanaka, Y. 2008. A rapid and easy multiresidue method for the determination of pesticide residues in vegetables, fruits, and cereals using liquid chromatography/tandem mass spectrometry. Journal of Aoac International 91(4): 871–883. Hernando, M.D.; Ferrer, C.; Ulazewska, M.; García-Reyes, J.F.; Molina-Díaz, A.; FernándezAlba, A.R. 2007. Application of high-performance

Pesticides: Herbicides and Fungicides

[154]

[155]

[156]

[157]

[158]

[159]

[160]

[161]

[162]

[163]

liquid chromatography–tandem mass spectrometry with a quadrupole/linear ion trap instrument for the analysis of pesticide residues in olive oil. Analytical and Bioanalytical Chemistry 389(6): 1815–1831. Banerjee, K.; Oulkar, D.P.; Dasgupta, S.; Patil, S.B.; Patil, S.H.; Savant, R.; Adsule, P.G. 2007. Validation and uncertainty analysis of a multiresidue method for pesticides in grapes using ethyl acetate extraction and liquid chromatographytandem mass spectrometry. Journal of Chromatography A 1173(1–2):98–109. Ferrer, I.; Fernandez-Alba, A.; Zweigenbaum, J.A.; Thurman, E.M. 2006. Exact-mass library for pesticides using a molecular-feature database. Rapid Communications in Mass Spectrometry 20(24):3659–3668. Ferrer, I.; Thurman, E.M. 2007. Multi-residue method for the analysis of 101 pesticides and their degradates in food and water samples by liquid chromatography/time-of-flight mass spectrometry. Journal of Chromatography A 1175(1): 24–37. Mezcua, M.; Malato, O.; Garcia-Reyes, J.F.; Molina-Díaz, A.; Fernandez-Alba, A.R. 2008. Accurate-mass databases for comprehensive screening of pesticide residues in food by fast liquid chromatography time-of-flight mass spectrometry. Analytical Chemistry 81(3):913–929. Mhaka, B.; Cukrowska, E.; Tse Sum Bui, B.; Ramström, O.; Haupt, K.; Tutu, H.; Chimuka, L. 2009. Selective extraction of triazine herbicides from food samples based on a combination of a liquid membrane and molecularly imprinted polymers. Journal of Chromatography A 1216(40): 6796–6801. Mondello, L.; Casilli, A.; Tranchida, P.Q.; Lo Presti, M.; Dugo, P.; Dugo, G. 2007. Comprehensive gas chromatography coupled to mass spectrometry for the separation of pesticides in a very complex matrix. Analytical and Bioanalytical Chemistry 389(6):1755–1763. Zhang, H.; Chen, Z.; Yang, G.; Wang, W.; Li, X.; Li, R.; Wu, Y. 2008. Microwave pretreatment and gas chromatography-mass spectrometry determination of herbicide residues in onion. Food Chemistry 108(1):322–328. Aramendía, M.A.; Borau, V.; Lafont, F.; Marinas, A.; Marinas, J.M.; Moreno, J.M.; Porras, J.M.; Urbano, F.J. 2006. Determination of diquat and paraquat in olive oil by ion-pair liquid chromatography-electrospray ionization mass spectrometry (MRM). Food Chemistry 97(1): 181–188. Schaner, A.; Konecny, J.; Luckey, L.; Hickes, H. 2007. Determination of chlorinated acid herbicides in vegetation and soil by liquid chromatography/electrospray-tandem mass spectrometry. Journal of AOAC International 90(5):1402– 1410. Chen, Z.; Zhang, H.; Liu, B.; Yang, G.; AboulEnein, H.Y.; Wang, W.; Ding, R.; Du, H.; Li, H. 2007. Determination of herbicide residues in

[164]

[165]

[166]

[167]

[168]

[169]

[170]

[171]

[172] [173]

[174]

191

garlic by GC–MS. Chromatographia 66(11/12): 887–891. Lin, C.H.; Lerch, R.N.; Garrett, H.E.; Li, Y.X.; George, M.F. 2007. Improved HPLC-MS/MS method for determination of isoxaflutole (balance) and its metabolites in soils and forage plants. Journal of Agricultural and Food Chemistry 55(10):3805–3815. Mou, R.X.; Chen, M.X.; Zhi, J.L. 2008. Simultaneous determination of 15 phenylurea herbicides in rice and corn using HPLC with fluorescence detection combined with UV decomposition and post-column derivatization. Journal of Chromatography B 875(2):437–443. You, J.; Zhang, H.; Zhang, H.; Yu, A.; Xiao, T.; Wang, Y.; Song, D. 2007. Determination of triazines in infant nutrient cereal-based foods by pressurized microwave-assisted extraction coupled with high-performance liquid chromatography-mass spectrometry. Journal of Chromatography B 856(1–2):278–284. Bichon, E.; Dupuis, M.; Le Bizec, B.; André, F. 2006. LC-ESI-MS/MS determination of phenylurea and triazine herbicides and their dealkylated degradation products in oysters. Journal of Chromatography B 838(2):96–106. Wang, S.; Xu, Y.; Pan, C.; Jiang, S.; Liu, F. 2007. Application of matrix solid-phase dispersion and liquid chromatography–mass spectrometry to fungicide residue analysis in fruits and vegetables. Analytical and Bioanalytical Chemistry 387(2): 673–685. Arrebola, F.J.; Martínez-Vidal, J.L.; GonzálezRodríguez, M.J.; Garrido-Frenich, A.; SánchezMorito, N. 2003. Reduction of analysis time in gas chromatography. Application of low-pressure gas chromatography–tandem mass spectrometry to the determination of pesticide residues in vegetables. Journal of Chromatography A 1005(1–2): 131–141. Ortelli, D.; Edder, P.; Corvi, C. 2004. Multiresidue analysis of 74 pesticides in fruits and vegetables by liquid chromatography–electrospray–tandem mass spectrometry. Analytica Chimica Acta 520(1–2):33–45. Walorczyk, S.; Gnusowski, B. 2006. Fast and sensitive determination of pesticide residues in vegetables using low-pressure gas chromatography with a triple quadrupole mass spectrometer. Journal of Chromatography A 1128(1–2): 236–243. Húsková, R.; Matisová, E.; Kirchner, M. 2008. Fast GC–MS pesticide multiresidue analysis of apples. Chromatographia 68(S):49–55. Viñas, P.; Campillo, N.; Aguinaga, N.; MartínezCastillo, N.; Hernández-Córdoba, M. 2008. Solidphase microextraction for the gas chromatography mass spectrometric determination of oxazole fungicides in malt beverages. Analytical and Bioanalytical Chemistry 391(4):1425–1431. de Melo Abreu, S.; Caboni, P.; Cabras, P.; Luigi Garau, V.; Alves, A. 2006. Validation and global uncertainty of a liquid chromatographic with

192

[175]

[176]

[177]

[178]

[179]

[180]

[181]

[182]

[183] [184]

Analysis of Endocrine Disrupting Compounds in Food

diode array detection method for the screening of azoxystrobin, kresoxim-methyl, trifloxystrobin, famoxadone, pyraclostrobin and fenamidone in grapes and wine. Analytica Chimica Acta 573–574:291–297. Shokrzadeh, M.; Saeedi Saravi, S.S. 2009. The investigation and measurement of residues of benomyl and mancozeb pesticides in shrub and nonshrub cucumbers sampled from different regions of Mazandaran province (Iran). Electronic Journal of Environmental, Agricultural and Food Chemistry 8(3):174–178. Ticha, J.; Hajslova, J.; Kovalczuk, T.; Jech, M.; Honzicek, J.; Kocourek, V.; Lansky, M.; Kloutvorova, J.; Falta, V. 2007. Safe apples for baby-food production: Survey of pesticide treatment regimes leaving minimum residues. Food Additives and Contaminants: Part A 24(6): 605–620. de Melo Abreu, S.; Correia, M.; Herbert, P.; Santos, L.; Alves, A. 2005. Screening of grapes and wine for azoxystrobin, kresoxim-methyl and trifloxystrobin fungicides by HPLC with diode array detection. Food Additives and Contaminants 22(6):549–556. Fernández Moreno, J.L.; Arrebola Liébanas, F.J.; Garrido Frenich, A.; Martínez Vidal, J.L. 2006. Evaluation of different sample treatments for determining pesticide residues in fat vegetable matrices like avocado by low-pressure gas chromatography–tandem mass spectrometry. Journal of Chromatography A 1111(1):97–105. da Silva, C.L.; de Lima, E.C.; Tavares, M.F.M. 2003. Investigation of preconcentration strategies for the trace analysis of multi-residue pesticides in real samples by capillary electrophoresis. Journal of Chromatography A 1014(1–2):109–116. Kodama, S.; Yamamoto, A.; Saitoh, Y.; Matsunaga, A.; Okamura, K.; Kizu, R.; Hayakawa, K. 2002. Enantioseparation of vinclozolin by γcyclodextrin-modified micellar electrokinetic chromatography. Journal of Agricultural and Food Chemistry 50(5):1312–1317. Stepan, R.; Ticha, J.; Hajslova, J.; Kovalczuk, T.; Kocourek, V. 2005. Baby food production chain: Pesticide residues in fresh apples and products. Food Additives and Contaminants 22(12): 1231–1242. Teixeira, M.J.; Aguiar, A.; Afonso, C.M.M.; Alves, A.; Bastos, M.M.S.M. 2004. Comparison of pesticides levels in grape skin and in the whole grape by a new liquid chromatographic multiresidue methodology. Analytica Chimica Acta 513(1):333–340. Ortelli, D.; Edder, P.; Corvi, C. 2005. Pesticide residues survey in citrus fruits. Food Additives and Contaminants 22(5):423–428. Patel, K.; Fussell, R.J.; Goodall, D.M.; Keely, B.J. 2004. Evaluation of large volume-difficult matrix introduction-gas chromatography-time of flightmass spectrometry (LV-DMI-GC-TOF-MS) for the determination of pesticides in fruit-based baby foods. Food Additives and Contaminants 21(7): 658–669.

[185] Juan-García, A.; Picó, Y.; Font, G. 2005. Capillary electrophoresis for analyzing pesticides in fruits and vegetables using solid-phase extraction and stir-bar sorptive extraction. Journal of Chromatography A 1073(1–2):229–236. [186] Özhan, G.; Alpertunga, B. 2008. Liquid chromatographic analysis of maneb and its main degradation product, ethylenethiouera, in fruit juice. Food Additives and Contaminants 25(8):961– 970. [187] Li, W.; Wu, Y.-J.; Qin, D.-M.; Ma, Y.; Sun, Y.-J.; Qiu, S.-P. 2008. A method for quantifying azoxystrobin residues in grapes and soil using GC with electron capture detection. Chromatographia 67(9–10):761–766. [188] Juan-García, A.; Font, G.; Picó, Y. 2005. Quantitative analysis of six pesticides in fruits by capillary electrophoresis-electrospray-mass spectrometry. Electrophoresis 26(7–8):1550– 1561. [189] Schurek, J.; Vaclavik, L.; Hooijerink, H.; Lacina, O.; Poustka, J.; Sharman, M.; Caldow, M.; Nielen, M.W.F.; Hajslova, J. 2008. Control of strobilurin fungicides in wheat using direct analysis in real time accurate time-of-flight and desorption electrospray ionization linear ion trap mass spectrometry. Analytical Chemistry 80(24):9567–9575. [190] Rodríguez, R.; Mañes, J.; Picó, Y. 2003. Off-line solid-phase microextraction and capillary electrophoresis mass spectrometry to determine acidic pesticides in fruits. Analytical Chemistry 75(3):452–459. [191] Martínez Vidal, J.L.; Fernández Moreno, J.L.; Arrebola Liébanas, F.J.; Garrido Frenich, A. 2007. Application of low-pressure gas chromatography/ tandem mass spectrometry to the determination of pesticide residues in tropical fruits. Journal of AOAC International 90(4):1146–1164. [192] Maeda, O.; Oikawa, C.; Shiomi, N.; Toriba, A.; Hayakawa, K. 2008. A clean-up method by photocatalysis for HPLC analysis of iprodione in dry basil. Analytical Sciences 24(8):1053–1055. [193] Hayama, T.; Takada, M. 2008. Simple and rapid method for the determination of ethylenebisdithiocarbamate fungicides in fruits and vegetables using liquid chromatography with tandem mass spectrometry. Analytical and Bioanalytical Chemistry 392(5):969–976. [194] Segura Carretero, A.; Cruces-Blanco, C.; Cortacero Ramírez, S.; Carrasco Pancorbo, A.; Fernández Gutiérrez, A. 2004. Application of micellar electrokinetic capillary chromatography to the analysis of uncharged pesticides of environmental impact. Journal of Agricultural and Food Chemistry 52(19):5791–5795. [195] Park, Y.-S.; Abd El-Aty, A.M.; Choi, J.-H.; Cho, S.-K.; Shin, D.-H.; Shim, J.-H. 2007. Pesticide multiresidue analysis in Panax ginseng (C.A. Meyer) by solid-phase extraction and gas chromatography with electron capture and nitrogen-phosphorus detection. Biomedical Chromatography 21(1):29–39. [196] Hernández-Borges, J.; Rodríguez-Delgado, M.Á.; García-Montelongo, F.J.; Cifuentes, A. 2004.

Pesticides: Herbicides and Fungicides

[197]

[198]

[199]

[200]

[201]

[202]

[203]

[204]

[205]

[206]

[207]

Highly sensitive analysis of multiple pesticides in foods combining solid-phase microextraction, capillary electrophoresis-mass spectrometry, and chemometrics. Electrophoresis 25(13):2065– 2076. González-Rodríguez, R.M.; Rial-Otero, R.; Cancho-Grande, B.; Simal-Gándara, J. 2008. Occurrence of fungicide and insecticide residues in trade samples of leafy vegetables. Food Chemistry 107(3):1342–1347. Scordino, M.; Sabatino, L.; Traulo, P.; Gagliano, G.; Gargano, M.; Pantò, V.; Gambino, Grazia L. 2008. LC/MS/MS detection of fungicide guazatine residues for quality assessment of commercial citrus fruit. European Food Research and Technology 227(5):1339–1347. Rawn, D.F.K.; Quade, S.C.; Sun, W.-F.; Fouguet, A.; Bélanger, A.; Smith, M. 2008. Captan residue reduction in apples as a result of rinsing and peeling. Food Chemistry 109(4):790–796. Shim, J.-H.; Abd El-Aty, A.M.; Choi, J.-H.; Kang, C.-A. 2007. Determination of field-incurred pyrimethanil residues in persimmon (Diospyros kaki Linn) by liquid chromatography. Biomedical Chromatography 21(12):1279–1283. Startin, J.R.; Hird, S.J.; Sykes, M.D. 2005. Determination of ethylenethiourea (ETU) and propylenethiourea (PTU) in foods by high performance liquid chromatography–atmospheric pressure chemical ionisation–medium-resolution mass spectrometry. Food Additives and Contaminants 22(3):245–250. Ochiai, N.; Sasamoto, K.; Kanda, H.; Nakamura, S. 2006. Fast screening of pesticide multiresidues in aqueous samples by dual stir bar sorptive extraction-thermal desorption-low thermal mass gas chromatography–mass spectrometry. Journal of Chromatography A 1130(1):83–90. Cengiz, M.F.; Certel, M.; Karakas, B.; Göçmen, H. 2007. Residue contents of captan and procymidone applied on tomatoes grown in greenhouses and their reduction by duration of a pre-harvest interval and post-harvest culinary applications. Food Chemistry 100(4):1611–1619. Yamamoto, A.; Miyamoto, I.; Kitagawa, M.; Moriwaki, H.I; Miyakoda, H.; Kawasaki, H.; Arakawa, R. 2009. Analysis of chlorothalonil by liquid chromatography/mass spectrometry using negative-ion atmospheric pressure photoionization. Analytical Sciences 25(5):693– 697. Nguyen, T.D.; Yu, J.E.; Lee, D.M.; Lee, G.-H. 2008. A multiresidue method for the determination of 107 pesticides in cabbage and radish using QuEChERS sample preparation method and gas chromatography mass spectrometry. Food Chemistry 110(1):207–213. Fenoll, J.; Ruiz, E.; Hellín, P.; Lacasa, A.; Flores, P. 2009. Dissipation rates of insecticides and fungicides in peppers grown in greenhouse and under cold storage conditions. Food Chemistry 113(3):727–732. Yuan, J.-T.; Liao, L.-F.; Xiao, X.-L.; He, B.; Gao, S.-Q. 2009. Analysis of malachite green and

[208]

[209] [210]

[211]

[212]

[213]

[214]

[215]

[216]

[217]

[218]

[219]

193

crystal violet in fish with bilinear model. Food Chemistry 113(4):1377–1383. Ticha, J.; Hajslova, J.; Jech, M.; Honzicek, J.; Lacina, O.; Kohoutkova, J.; Kocourek, V.; Lansky, M.; Kloutvorova, J.; Falta, V. 2008. Changes of pesticide residues in apples during cold storage. Food Control 19(3):247–256. Knezevic, Z.; Serdar, M. 2009. Screening of fresh fruit and vegetables for pesticide residues on Croatian market. Food Control 20(4):419–422. Malik, A.K.; Sharma, V.; Sharma, V.K.; Rao, A.L.J. 2004. Column preconcentration and spectrophotometric determination of ziram and zineb in commercial samples and foodstuffs using (1,2′-pyridylazo)-2-naphthol(pan)naphthalene as adsorbate. Journal of Agricultural and Food Chemistry 52(26):7763–7767. Rial-Otero, R.; Arias-Estévez, M.; López-Periago, E.; Cancho-Grande, B.; Simal-Gándara, J. 2005. Variation in concentrations of the fungicides tebuconazole and dichlofluanid following successive applications to greenhouse-grown lettuces. Journal of Agricultural and Food Chemistry 53(11):4471–4475. Schirra, M.; D’Aquino, S.; Palma, A.; Marceddu, S.; Angioni, A.; Cabras, P.; Scherm, B.; Migheli, Q. 2005. Residue level, persistence, and storage performance of citrus fruit treated with fludioxonil. Journal of Agricultural and Food Chemistry 53(17):6718–6724. Navarro, S.; Pérez, G.; Vela, N.; Mena, L.; Navarro, G. 2005. Behavior of myclobutanil, propiconazole, and nuarimol residues during lager beer brewing. Journal of Agricultural and Food Chemistry 53(22):8572–8579. Tsiropoulos, N.G.; Miliadis, G.E.; Likas, D.T.; Liapis, K. 2005. Residues of spiroxamine in grapes following field application and their fate from vine to wine. Journal of Agricultural and Food Chemistry 53(26):10091–10096. Lentza-Rizos, C.; Avramides, E.J.; Kokkinaki, K. 2006. Residues of azoxystrobin from grapes to raisins. Journal of Agricultural and Food Chemistry 54(1):138–141. Rabølle, M.; Spliid, N.H.; Kristensen, K.; Kudsk, P. 2006. Determination of fungicide residues in field-grown strawberries following different fungicide strategies against gray mold (Botrytis cinerea). Journal of Agricultural and Food Chemistry 54(3):900–908. Andersen, W.C.; Turnipseed, S.B.; Roybal, J.E. 2006. Quantitative and confirmatory analyses of malachite green and leucomalachite green residues in fish and shrimp. Journal of Agricultural and Food Chemistry 54(13):4517–4523. D’Aquino, S.; Schirra, M.; Palma, A.; Angioni, A.; Cabras, P.; Migheli, Q. 2006. Residue levels and effectiveness of pyrimethanil vs imazalil when using heated postharvest dip treatments for control of penicillium decay on citrus fruit. Journal of Agricultural and Food Chemistry 54(13):4721–4726. Kolbe, N.; Andersson, J.T. 2006. Simple and sensitive determination of o-phenylphenol in citrus

194

[220]

[221]

[222]

[223]

[224]

[225]

[226]

[227]

[228]

[229]

Analysis of Endocrine Disrupting Compounds in Food

fruits using gas chromatography with atomic emission or mass spectrometric detection. Journal of Agricultural and Food Chemistry 54(16): 5736–5741. Dreassi, E.; Zizzari, A.T.; Zanfini, A.; Corbini, G.; Botta, M. 2007. LC/ESI/MS method for the quantitative detection of guazatine residues in cereals. Journal of Agricultural and Food Chemistry 55(17):6850–6856. Gilbert-López, B.; García-Reyes, J.F.; Mezcua, M.; Molina-Díaz, A.; Fernández-Alba, A.R. 2007. Determination of postharvest fungicides in fruit juices by solid-phase extraction followed by liquid chromatography electrospray time-of-flight mass spectrometry. Journal of Agricultural and Food Chemistry 55(26):10548–10556. Garau, V.L.; de Melo Abreu, S.; Caboni, P.; Angioni, A.; Alves, A.; Cabras, P. 2009. Residuefree wines: Fate of some quinone outside inhibitor (qoi) fungicides in the winemaking process. Journal of Agricultural and Food Chemistry 57(6):2329–2333. Zhang, K.; Wong, J.W.; Hayward, D.G.; Sheladia, P.; Krynitsky, A.J.; Schenck, F.J.; Webster, M.G.; Ammann, J.A.; Ebeler, S.E. 2009. Multiresidue pesticide analysis of wines by dispersive solidphase extraction and ultrahigh-performance liquid chromatography-tandem mass spectrometry. Journal of Agricultural and Food Chemistry 57(10):4019–4029. Maity, A.; Mukherjee, I. 2004. Liquid chromatographic determination of iprovalicarb in cabbage and soil. Journal of AOAC International 87(1): 157–161. Navickiene, S.; Ribeiro, M.L. 2004. Determination of fluquinconazole, pyrimethanil, and clofentezine residues in fruits by liquid chromatography with ultraviolet detection. Journal of AOAC International 87(2):435–438. Albero, B.; Sánchez-Brunete, C.; Tadeo, J.L. 2004. Determination of thiabendazole in orange juice and rind by liquid chromatography with fluorescence detection and confirmation by gas chromatography/mass spectrometry after extraction by matrix solid-phase dispersion. Journal of AOAC International 87(3):664–670. Sannino, A. 2004. Evaluation of a method based on liquid chromatography/electrospray tandem mass spectrometry for analyzing eight triazolic and pyrimidine fungicides in extracts of processed fruits and vegetables. Journal of AOAC International 87(4):991–996. Schermerhorn, P.G.; Golden, P.E.; Krynitsky, A.J.; Leimkuehler, W.M. 2005. Determination of 22 triazole compounds including parent fungicides and metabolites in apples, peaches, flour, and water by liquid chromatography/tandem mass spectrometry. Journal of AOAC International 88(5):1491–1502. Tsiropoulos, N.G.; Liapis, K.; Likas, D.T.; Miliadis, G.E. 2005. Determination of spiroxamine residues in grapes, must, and wine by gas chromatography/ion trap-mass spectrometry.

[230]

[231]

[232]

[233]

[234]

[235]

[236]

[237]

[238]

[239]

Journal of AOAC International 88(6):1834– 1839. Barreda, M.; López, F.J.; Villarroya, M.; Beltran, J.; García-Baudín, J.M.; Hernández, F. 2006. Residue determination of captan and folpet in vegetable samples by gas chromatography/negative chemical ionization–mass spectrometry. Journal of AOAC International 89(4):1080– 1087. Aulakh, J.S.; Fekete, A.; Malik, A.K.; Mahajan, R.K.; Schmitt-Kopplin, P. 2007. Capillary electrophoretic-ultraviolet method for the separation and estimation of zineb, maneb, and ferbam in food samples. Journal of AOAC International 90(3): 834–837. Hu, J.-Y.; Zhang, Y.-C.; Yan, H. 2008. Determination of flumorph residues in vegetables, soil, and natural water by solid-phase extraction cleanup and high-performance liquid chromatography with UV detection. Journal of AOAC International 91(6):1459–1466. Yoshioka, N.; Akiyama, Y.; Teranishi, K. 2004. Rapid simultaneous determination of o-phenylphenol, diphenyl, thiabendazole, imazalil and its major metabolite in citrus fruits by liquid chromatography-mass spectrometry using atmospheric pressure photoionization. Journal of Chromatography A 1022(1–2):145–150. Melo, L.F.C.; Collins, C.H.; Jardim, I.C.S.F. 2004. New materials for solid-phase extraction and multiclass high-performance liquid chromatographic analysis of pesticides in grapes. Journal of Chromatography A 1032(1–2):51–58. Sannino, A.; Bolzoni, L.; Bandini, M. 2004. Application of liquid chromatography with electrospray tandem mass spectrometry to the determination of a new generation of pesticides in processed fruits and vegetables. Journal of Chromatography A 1036(2):161–169. Zamora, T.; Pozo, O.J.; López, F.J.; Hernández, F. 2004. Determination of tridemorph and other fungicide residues in fruit samples by liquid chromatography–electrospray tandem mass spectrometry. Journal of Chromatography A 1045(1–2): 137–143. Valle, L.; Díaz, C.; Zanocco, A.L.; Richter, P. 2005. Determination of the sum of malachite green and leucomalachite green in salmon muscle by liquid chromatography–atmospheric pressure chemical ionisation-mass spectrometry. Journal of Chromatography A 1067(1–2):101–105. Nozal, M..J.; Bernal, J.L.; Jiménez, J.J.; Martín, M.T.; Bernal, J. 2005. Determination of azolic fungicides in wine by solid-phase extraction and high-performance liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry. Journal of Chromatography A 1076(1–2):90–96. Thurman, E.M.; Ferrer, I.; Zweigenbaum, J.A.; García-Reyes, J.F.; Woodman, M.; FernándezAlba, A.R. 2005. Discovering metabolites of postharvest fungicides in citrus with liquid chromatography/time-of-flight mass spectrometry

Pesticides: Herbicides and Fungicides

[240]

[241]

[242]

[243]

[244]

[245]

[246]

[247]

[248]

[249]

[250]

and ion trap tandem mass spectrometry. Journal of Chromatography A 1082(1):71–80. Trösken, E.R.; Bittner, N.; Völkel, W. 2005. Quantitation of 13 azole fungicides in wine samples by liquid chromatography–tandem mass spectrometry. Journal of Chromatography A 1083(1–2):113–119. Mitrowska, K.; Posyniak, A.; Zmudzki, J. 2005. Determination of malachite green and leucomalachite green in carp muscle by liquid chromatography with visible and fluorescence detection. Journal of Chromatography A 1089(1–2): 187–192. Patel, K.; Fussell, R.J.; Goodall, D.M.; Keely, B.J. 2003. Analysis of pesticide residues in lettuce by large volume–difficult matrix introduction– gas chromatography–time of flight–mass spectrometry (LV-DMI-GC-TOF-MS). The Analyst 128(10):1228–1231. Cajka, T.; Hajslova, J. 2004. Gas chromatography–high-resolution time-of-flight mass spectrometry in pesticide residue analysis: Advantages and limitations. Journal of Chromatography A 1058(1–2):251–261. Hercegová, A.; Dömötörová, M.; Matisová, E.; Kirchner, M.; Otrekal, R.; Stefuca,V. 2005. Fast gas chromatography with solid phase extraction clean-up for ultratrace analysis of pesticide residues in baby food. Journal of Chromatography A 1084(1–2):46–53. Kirchner, M.; Matisová, E.; Otrekal, R.; Hercegová, A.; de Zeeuw, J. 2005. Search on ruggedness of fast gas chromatography–mass spectrometry in pesticide residues analysis. Journal of Chromatography A 1084(1–2):63–70. Cajka, T.; Hajslova, J.; Lacina, O.; Mastovska, K.; Lehotay, S.J. 2008. Rapid analysis of multiple pesticide residues in fruit-based baby food using programmed temperature vaporiser injection– low-pressure gas chromatography–high-resolution time-of-flight mass spectrometry. Journal of Chromatography A 1186(1–2):281–294. Sasamoto, K.; Ochiai, N.; Kanda, H. 2007. Dual low thermal mass gas chromatography–mass spectrometry for fast dual-column separation of pesticides in complex sample. Talanta 72(5): 1637–1643. Dallüge, J.; van Rijn, M.; Beens, J.; Vreuls, R.J.J.; Brinkman, U.A.Th. 2002. Comprehensive twodimensional gas chromatography with time-offlight mass spectrometric detection applied to the determination of pesticides in food extracts. Journal of Chromatography A 965(1–2): 207–217. Zrostlíková, J.; Hajšlová, J.; Cajka, T. 2002. Evaluation of two-dimensional gas chromatography–time-of-flight mass spectrometry for the determination of multiple pesticide residues in fruit. Journal of Chromatography A 1019(1–2): 173–186. Hernández-Borges, J.; Cifuentes, A.; GarcíaMontelongo, F.J.; Rodríguez-Delgado, M.Á. 2005. Combining solid-phase microextraction and

[251]

[252]

[253]

[254]

[255]

[256]

[257]

[258]

[259]

[260]

195

on-line preconcentration-capillary electrophoresis for sensitive analysis of pesticides in foods. Electrophoresis 26(4–5):980–989. Abreu, S.; Caboni, P.; Cabras, P.; Alves, A.; Garau, V.L. 2006. A comparison of a gas chromatographic with electron-capture detection and a gas chromatographic with mass spectrometric detection screening methods for the analysis of famoxadone in grapes and wines. Journal of Chromatography A 1103(2):362–367. Hernández, F.; Pozo, O.J.; Sancho, J.V.; Bijlsma, L.; Barreda, M.; Pitarch, E. 2006. Multiresidue liquid chromatography tandem mass spectrometry determination of 52 non gas chromatographyamenable pesticides and metabolites in different food commodities. Journal of Chromatography A 1109(2):242–252. Singh, S.B.; Foster, G.D.; Khan, S.U. 2007. Determination of thiophanate methyl and carbendazim residues in vegetable samples using microwave-assisted extraction. Journal of Chromatography A 1148(2):152–157. Likas, D.T.; Tsiropoulos, N.G.; Miliadis, G.E. 2007. Rapid gas chromatographic method for the determination of famoxadone, trifloxystrobin, and fenhexamid residues in tomato, grape, and wine samples. Journal of Chromatography A 1150(1–2):208–214. Juan-García, A.; Font, G.; Picó, Y. 2007. On-line preconcentration strategies for analyzing pesticides in fruits and vegetables by micellar electrokinetic chromatography. Journal of Chromatography A 1153(1–2):104–113. Wu, X.; Zhang, G.; Wu, Y.; Hou, X.; Yuan, Z. 2007. Simultaneous determination of malachite green, gentian violet and their leuco-metabolites in aquatic products by high-performance liquid chromatography–linear ion trap mass spectrometry. Journal of Chromatography A 1172(2): 121–126. Ravelo-Pérez, L.M.; Hernández-Borges, J.; Borges-Miquel, T.M.; Rodríguez-Delgado, M.Á. 2008. Pesticide analysis in tomatoes by solidphase microextraction and micellar electrokinetic chromatography. Journal of Chromatography A 1185(1):151–154. Kirchner, M.; Húsková, R.; Matisová, E.; Mocák, J. 2008. Fast gas chromatography for pesticide residues analysis using analyte protectants. Journal of Chromatography A 1186(1–2): 271–280. Banerjee, K.; Patil, S.H.; Dasgupta, S.; Oulkar, D.P.; Patil, S.B.; Savant, R.; Adsule, P.G. 2008. Optimization of separation and detection conditions for the multiresidue analysis of pesticides in grapes by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry. Journal of Chromatography A 1190(1–2): 350–357. González-Rodríguez, R.M.; Rial-Otero, R.; Cancho-Grande, B.; Simal-Gándara, J. 2008. Determination of 23 pesticide residues in leafy vegetables using gas chromatography–ion trap

196

[261]

[262]

[263]

[264]

[265]

[266]

[267]

[268]

[269]

[270]

[271]

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mass spectrometry and analyte protectants. Journal of Chromatography A 1196–1197: 100–109. Viñas, P.; Campillo, N.; Martínez-Castillo, N.; Hernández-Córdoba, M. 2009. Method development and validation for strobilurin fungicides in baby foods by solid-phase microextraction gas chromatography–mass spectrometry. Journal of Chromatography A 1216(1–2):140–146. González-Rodríguez, R.M.; Cancho-Grande, B.; Simal-Gándaram, J. 2009. Multiresidue determination of 11 new fungicides in grapes and wines by liquid–liquid extraction/clean-up and programmable temperature vaporization injection with analyte protectants/gas chromatography/ion trap mass spectrometry. Journal of Chromatography A 1216(32):6033–6042. Lee, K.-C.; Wu, J.-L.; Cai, Z. 2006. Determination of malachite green and leucomalachite green in edible goldfish muscle by liquid chromatography– ion trap mass spectrometry. Journal of Chromatography B 843(2):247–251. Halme, K.; Lindfors, E.; Peltonen, K. 2007. A confirmatory analysis of malachite green residues in rainbow trout with liquid chromatography– electrospray tandem mass spectrometry. Journal of Chromatography B 845(1):74–79. Michel, M.; Gnusowski, B.; Buszewski, B. 2006. Comparison of various extraction techniques to determine fungicide residue in wheat grain. Journal of Liquid Chromatography & Related Technologies 29(2):247–261. Cho, S.-K.; Abd El-Aty, A.M.; Choi, J.-H.; Jeong, Y.-M.; Shin, H.-C.; Chang, B.-J.; Lee, C.; Shim, J.-H. 2008. Effectiveness of pressurized liquid extraction and solvent extraction for the simultaneous quantification of 14 pesticide residues in green tea using GC. Journal of Separation Science 31(10):1750–1760. Khummueng, W.; Morrison, P.; Marriott, P.J. 2008. Dual NPD/ECD detection in comprehensive two dimensional gas chromatography for multiclass pesticide analysis. Journal of Separation Science 31(19):3404–3415. Karthika, C.; Sachi, P.J.N. 2008. Gas chromatographic method for the determination of hexaconazole residues in black tea. Journal of Zhejiang University SCIENCE B 9(2):160–164. XueMei, W.; HaiLong, W.; JinFang, N.; YuanNa, L.; YongJie, Y.; RuQin, Y. 2008. Rapid determination of thiabendazole in orange extract using excitation-emission matrix fluorescence and second-order calibration based on alternating trilinear decomposition/alternating normalizationweighted error algorithms. Science in China Series B: Chemistry 51(8):729–735. Amer, M.M.; Shehata, M.A.; Lotfy, H.M.; Monir, H.H. 2007. Determination of tetraconazole and diniconazole fungicide residues in tomatoes and green beans by capillary gas chromatography. Yakugaku Zasshi 127(6):993–999. Khay, S.; Abd El-Aty, A.M.; Choi, J.-H.; Shim, J.-H. 2008. Analysis of residual triflumizole, an imidazole fungicide, in apples, pears,

[272]

[273]

[274]

[275]

[276]

[277]

[278]

[279]

[280]

[281]

[282]

[283]

and cucumbers using high performance liquid chromatography. Toxicological Research 24(1): 87–91. Montes, R.; Rodríguez, I.; Ramil, M.; Rubí, E.; Cela, R. 2009. Solid-phase extraction followed by dispersive liquid–liquid microextraction for the sensitive determination of selected fungicides in wine. Journal of Chromatography A 1216(29): 5459–5466. Harrold, J.A.; Sykes, M.D.; Goodrum, M.; Fussell, R.J. 2004. Determination of triforine using highperformance liquid chromatography with tandem mass spectrometry. Rapid Communications in Mass Spectrometry 18(7):775–779. Crnogorac, G.; Schwack, W. 2007. Determination of dithiocarbamate fungicide residues by liquid chromatography/mass spectrometry and stable isotope dilution assay. Rapid Communications in Mass Spectrometry 21(24):4009–4016. Crnogorac, G.; Schmauder, S.; Schwack, W. 2008. Trace analysis of dithiocarbamate fungicide residues on fruits and vegetables by hydrophilic interaction liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 22(16):2539–2546. Oliva, J.; Payá, P.; Cámara, M.Á.; Barba, A. 2007. Removal of famoxadone, fluquinconazole and trifloxystrobin residues in red wines: Effect of clarification and filtration processes. Journal of Environmental Science and Health Part B 42(7):775–781. Blasco, C.; Font, G.; Mañes, J.; Picó, Y. 2003. Solid-phase microextraction liquid chromatography/tandem mass spectrometry to determine postharvest fungicides in fruits. Analytical Chemistry 75(14):3606–3615. Kaltsonoudis, C.K.; Lamari, F.N.; Prousalis, K.P.; Karamanos, N.K.; Tsegenidis, T. 2003. Analysis of carbendazim and thiabendazole in lemons by CE. Chromatographia 57(3–4):181–184. Michel, M.; Buszewski, B. 2002. HPLC determination of pesticide residue isolated from food matrices. Journal of Liquid Chromatography & Related Technologies 25(13–15):2293–2306. Hengel, M.J.; Shibamoto, T. 2002. Gas chromatographic-mass spectrometric method for the analysis of dimethomorph fungicide in dried hops. Journal of Agricultural and Food Chemistry 48(12):5824–5828. Schirra, M.; Cabras, P.; Angioni, A.; D’Hallewin, G.; Pala, M. 2002. Residue uptake and storage responses of tarocco blood oranges after preharvest thiabendazole spray and postharvest heat treatment. Journal of Agricultural and Food Chemistry 50(8):2293–2296. Kodama, S.; Yamamoto, A.; Ohura, T.; Matsunaga, A.; Kanbe, T. 2003. Enantioseparation of imazalil residue in orange by capillary electrophoresis with 2-hydroxypropyl-β-cyclodextrin as a chiral selector. Journal of Agricultural and Food Chemistry 51(21):6128–6131. Blasco, C.; Picó, Y.; Font, G. 2002. Monitoring of five postharvest fungicides in fruit and vegetables by matrix solid-phase dispersion and liquid chro-

Pesticides: Herbicides and Fungicides

[284]

[285]

[286]

[287]

[288]

[289]

[290]

[291]

[292]

[293]

[294]

matography/mass spectrometry. Journal of AOAC International 85(3):704–711. Sreedhar, N.Y.; Babu, T.R.; Samatha, K.; Sujatha, D.; Thriveni, T. 2002. Differential pulse polarographic determination of procymidone in formulations and wine. Journal of AOAC International 85(3):731–735. Hernández, F.; Sancho, J.V.; Pozo, Ó.J.; Villaplana, C.; Ibáññez, M.; Grimalt, S. 2003. Rapid determination of fosetyl-aluminum residues in lettuce by liquid chromatography/electrospray tandem mass spectrometry. Journal of AOAC International 86(4):832–838. Fernández González, C.; Rial Otero, R.; Cancho Grande, B.; Simal Gándara, J. 2003. Determination of fungicide residues in white grapes for winemaking by gas chromatography with mass spectrometric detection and assessment of matrix effects. Journal of AOAC International 86(5): 1008–1014. Rial Otero, R.; Yagüe Ruiz, C.; Cancho Grande, B.; Simal Gándara, J. 2002. Solid-phase microextraction–gas chromatographic–mass spectrometric method for the determination of the fungicides cyprodinil and fludioxonil in white wines. Journal of Chromatography A 942(1–2):41–52. Blasco, C.; Picó, Y.; Mañes, J.; Font, G. 2002. Determination of fungicide residues in fruits and vegetables by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry. Journal of Chromatography A 947(2): 227–235. Rodríguez, R.; Picó, Y.; Font, G.; Mañes, J. 2002. Analysis of thiabendazole and procymidone in fruits and vegetables by capillary electrophoresis– electrospray mass spectrometry. Journal of Chromatography A 949(1–2):359–366. Navarro, M.; Picó, Y.; Marín, R.; Mañes, J. 2002. Application of matrix solid-phase dispersion to the determination of a new generation of fungicides in fruits and vegetables. Journal of Chromatography A 968(1–2):201–209. Navalón, A.; Prieto, A.; Araujo, L.; Vílchez, J.L. 2002. Determination of pyrimethanil and kresoxim-methyl in green groceries by headspace solid-phase microextraction and gas chromatography–mass spectrometry. Journal of Chromatography A 975(2):355–360. Rial Otero, R.; Cancho Grande, B.; Simal Gándara, J. 2003. Multiresidue method for fourteen fungicides in white grapes by liquid–liquid and solid-phase extraction followed by liquid chromatography–diode array detection. Journal of Chromatography A 992(1–2):121–131. Millán, S.; Sampedro, M.C.; Unceta, N.; Goicolea, M.A.; Rodríguez, E.; Barrio, R.J. 2003. Coupling solid-phase microextraction and high-performance liquid chromatography for direct and sensitive determination of halogenated fungicides in wine. Journal of Chromatography A 995(1–2): 135–142. Kvasnicka, F.; Dobiás, J.; KlaudisováChudácková, K. 2003. Determination of imazalil

[295]

[296]

[297]

[298]

[299]

[300]

[301]

[302]

[303]

[304]

197

by on-line coupled capillary isotachophoresis with capillary zone electrophoresis. Central European Journal of Chemistry 1(1):91–97. Hayasaka, Y.; MacNamara, K.; Baldock, G.A.; Taylor, R.L.; Pollnitz, A.P. 2003. Application of stir bar sorptive extraction for wine analysis. Analytical and Bioanalytical Chemistry 375(7): 948–955. Vryzas, Z.; Papadakis, E.N.; PapadopoulouMourkidou, E. 2002. Microwave-assisted extraction (MAE)-acid hydrolysis of dithiocarbamates for trace analysis in tobacco and peaches. Journal of Agricultural and Food Chemistry 50(8): 2220–2226. Walorczyk, S. 2008. Development of a multiresidue method for the determination of pesticides in cereals and dry animal feed using gas chromatography–tandem quadrupole mass spectrometry II. Improvement and extension to new analytes. Journal of Chromatography A 1208(1–2): 202–214. Zambonin, C.G.; Cilenti, A.; Palmisano, F. 2002. Solid-phase microextraction and gas chromatography–mass spectrometry for the rapid screening of triazole residues in wine and strawberries. Journal of Chromatography A 967(2):255–260. Hiemstra, M.; de Kok, A. 2002. Determination of propamocarb in vegetables using polymer-based high-performance liquid chromatography coupled with electrospray mass spectrometry. Journal of Chromatography A 972(2):231–239. Taylor, M.J.; Hunter, K.; Hunter, K.B.; Lindsay, D.; Le Bouhellec, S. 2002. Multi-residue method for rapid screening and confirmation of pesticides in crude extracts of fruits and vegetables using isocratic liquid chromatography with electrospray tandem mass spectrometry. Journal of Chromatography A 982(2):225–236. Juan-García, A.; Mañes, J.; Font, G.; Picó, Y. 2004. Evaluation of solid-phase extraction and stir-bar sorptive extraction for the determination of fungicide residues at low-μg kg−1 levels in grapes by liquid chromatography–mass spectrometry. Journal of Chromatography A 1050(2): 119–127. Ravelo-Pérez, L.M.; Hernández-Borges, J.; Borges-Miquel, T.M.; Rodríguez-Delgado, M.Á. 2007. Multiple pesticide analysis in wine by MEKC combined with solid-phase microextraction and sample stacking. Electrophoresis 28(22):4072–4081. Mercader, J.V.; Abad-Fuentes, A. 2009. Monoclonal antibody generation and direct competitive enzyme-linked immunosorbent assay evaluation for the analysis of the fungicide fenhexamid in must and wine. Journal of Agricultural and Food Chemistry 57(12):5129–5135. Húsková, R.; Matisová, E.; Hrouzková, S.; Svorc, L. 2009. Analysis of pesticide residues by fast gas chromatography in combination with negative chemical ionization mass spectrometry. Journal of Chromatography A 1216(35):6326– 6334.

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[305] Kurz, M.H.S.; Gonçalves, F.F.; Adaime, M.B.; da Costa, I.F.D.; Primel, E.G.; Zanella, R. 2008. A gas chromatographic method for the determination of the fungicide chlorothalonil in tomatoes and cucumbers and its application to dissipation studies in experimental greenhouses. Journal of the Brazilian Chemical Society 19(6):1129– 1135. [306] Navickiene, S.; Ribeiro, M.L. 2005. An alternative LC-UV procedure for the determination of prochloraz residues in fruits. Journal of the Brazilian Chemical Society 16(2):157–162.

[307] Sannino, A.; Bandini, M. 2005. Determination of fludioxonil and famoxadone in processed fruits and vegetables by liquid chromatography/electrospray tandem mass spectrometry. Journal of AOAC International 88(6):1822–1826. [308] Lüthje, K.; Hyötyláinen, T.; Rautiainen-Rámá, M.; Riekkola, M.-L. 2005. Pressurised hot water extraction–microporous membrane liquid– liquid extraction coupled on-line with gas chromatography–mass spectrometry in the analysis of pesticides in grapes. The Analyst 130(1): 52–58.

Chapter 6 Pesticides: Organophosphates Juan F. García-Reyes, Bienvenida Gilbert-López, and Antonio Molina-Díaz

Introduction Since 1990, intensive agriculture has highlighted the need for procedures to study the impact of pollutants in crops and foodstuffs. Among them, organophosphate compounds (OPCs) are a diverse group of chemicals used in both domestic and industrial settings. Examples of organophosphates include insecticides (malathion, diazinon), nerve gases (sarin, VX, soman), antihelmintics (trichlorfon), etc. OPCs include some of the most toxic chemicals used in agriculture. Acephate, chlorpyrifos, dimethoate, diazinon, terbufos, malathion, and parathion are among the more widely used organophosphorus (OP) pesticides (Figure 6.1). These agrochemicals are esters, amides, or simple derivatives of phosphoric and thiophosphoric acids. Some of the less toxic compounds are used as systemic insecticides in animals against internal and external parasites. OPCs vary greatly in their toxic capabilities and have the advantage over other types of insecticides in that they produce little or no tissue residues. All have a cumulative effect, with chronic exposure causing progressive inhibition of cholinesterase. Liquid OPCs are absorbed readily by all routes, although malathion, which is the least toxic of these chemi-

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

cals, is only slightly absorbed through the skin. Organophosphate toxicity is due to the ability of these compounds to inhibit acetylcholinesterase at cholinergic junctions of the nervous system (Lord and Potter 1950). These junctions include postganglionic parasympathetic neuroeffector junctions (sites of muscarinic activity), autonomic ganglia and the neuromuscular junctions (sites of nicotinic activity), and certain synapses in the central nervous system. Many organophosphate pesticides and their metabolites have been shown to be carcinogenic endocrine disruptors (Kitamura et al. 2006). Endocrine-disrupting chemicals are compounds that alter the normal functioning of the endocrine system, potentially causing disease or deformity in organisms and their offspring. As an overview, Table 6.1 shows the endocrine-disrupting properties of the main organophosphate pesticides (McKinlay et al. 2008). The two main endocrine disruption mechanisms of organophosphorus pesticides are (1) the prevention of thyroid hormone-receptor binding and (2) the increase of the expression of estrogen-responsive genes. Cabello et al. (2001) were the first to demonstrate that organophosphates can cause mammary tissue carcinogenesis in rats. Concentrations of malathion as low as 10 nM have been shown to damage normal cells in vitro (Cabello et al. 2003). An investigation of gene expression in different strains of human mammary cells exposed to malathion in vitro (Bustos 199

200

Analysis of Endocrine Disrupting Compounds in Food

H3 C

Cl

O

O

N H

P

OCH3 SCH 3

S H3 C

P

O O

acephate

Cl

N

S H 3CO

O

S OCH3

dimethoate

chlorpyrifos

H3 C

O

CH3

O P

O

N

(H 3C)3C

S

P

S

CH3 CH3

terbufos

CH O 3

S

O

CH3

P

O

CH3

S

O O

N

diazinon

H 3CO H 3CO

S CH 3

S

CH

O

Cl

H3C H 3C

H N

P

NO 2

S H3 C

P

O O

O

H 3C

O

parathion

malathion Figure 6.1. Structure of selected organophosphorus pesticides.

et al. 2001) found that the expression of genes associated with the progression of the cell cycle, including those responsible for the preservation of chromosomal integrity, was suppressed and the expression of genes associated with carcinogenesis and steroid metabolism increased. Other organophosphates have been shown to carry similar risks. For this reason, there is a need to control and characterize the exposure to organophosphorus pesticides, and this can be accomplished with effective analytical methodologies. The determination of OP pesticides has been performed by use of, for example, capillary electrophoresis (Wuilloud et al. 2005), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) (Shu et al. 2006), desorption electrospray ionization mass spectrometry (DESI-MS) (Hagan et al. 2008), ion mobility time-of-flight MS (IMSTOFMS) (Steiner et al. 2005), and many

more techniques. In contrast to these rather unusual methods, gas chromatography and liquid chromatography coupled to mass spectrometry represents the most commonly used methods for the determination of OP pesticides in food and environmental samples. However, these techniques are to some extent time consuming and expensive, and they demand a qualified and experienced staff and cannot be used for continuous monitoring. For this reason, several fast approaches have been proposed as alternatives, including biological techniques such as immunoassays or biosensors based on the inhibition of cholinesterase activity (Trojanowicz and Hitchman 1996). This chapter intends to provide an insight into the more commonly used methodologies for the analysis of OP pesticides in food. We devote special attention only to those methodologies that have been proposed and evalu-

Pesticides: Organophosphates

201

Table 6.1. Endocrine-disrupting properties of selected organophosphorus pesticides. Pesticide

Description

Endocrine-Disrupting Effects

Acephate

Systemic insecticide

Chlorfenvinphos Chlorpyrifos methyl Dichlorvos

Insecticide Insecticide Insecticide, acaricide

Dimethoate

Insecticide, acaricide

Fenitrothion

Insecticide

Glyphosate

Herbicide

Malathion

Insecticide, acaricide

Methomyl

Insecticide, acaricide

Parathion

Insecticide, acaricide

Disrupts hormone expression in the hypothalamus Weak estrogen mimic Antagonizes androgen activity Weak androgen-receptor antagonist Disrupts the action of the thyroid hormones. Increases the blood concentration of insulin and decreases the blood concentration of luteinizing hormone Antagonizes the action of androgens by binding to their receptors. Also inhibits the action of estrogen Disrupts the action of aromatase, preventing the production of estrogens Inhibits catecholamine secretion, binds to thyroid hormone receptors Weakly promotes aromatase activity, increasing estrogen production Inhibits catecholamine secretion, increases nocturnal synthesis of melatonin, causes gonadotrophic hormone inhibition

References Singh 2002 Vinggaard et al. 2005 Kang et al. 2004 Andersen et al. 2002 Mahjoubi-Samet et al. 2005; Boujelben et al. 2005 Okubo et al. 2004

Richard et al. 2005 Cocco 2002 Andersen et al. 2002 Cocco 2002

Adapted from McKinlay et al. (2008).

ated in detail for OP pesticide analysis. The main chromatographic and nonchromatographic approaches are described in detail with an emphasis on the sample treatment step, which is the main bottleneck on the overall throughput of the described methods.

Sample treatment techniques used for organophosphorus pesticide analysis Regardless of the detection method, a sample preparation step is always required before pesticide residue analysis in food. According to the literature, a variety of sample preparation techniques have been used for the extraction of OP pesticides from food matrices. Table 6.2 shows an overview of the different methods proposed for the sample treatment

and extraction of OP pesticides from foodstuffs. Liquid partitioning with organic solvents and solid-phase extraction are the more widely used sample treatment techniques for pesticide extraction in foodstuffs. The main sample treatment approach for OP pesticide analysis in fruits and vegetables is roughly based on liquid partitioning with organic solvents such as ethyl acetate, acetonitrile, and dichloromethane, usually followed by a solid-phase extraction cleanup step. Of particular interest is the QuEChERS method, an acronym of quick, easy, cheap, effective, rugged and safe, first described by Anastassiades and Lehotay (Anastassiades et al. 2003). This method is based on liquid partitioning with acetonitrile followed by a cleanup step by dispersive solid-phase extraction with primary secondary amine as sorbent

Table 6.2. Selected sample preparation methods for OP pesticide analysis. Targeted Analytes

Sample/Matrix

Chlorpyrifos methyl, diazinon, fonofos, phenthoate, phosalone, and pirimiphos ethyl

honey

Methidathion, azinphos-methyl, malathion, pirimiphosmethyl, etrimfos, pyraclofos, and phosalone Chlorpyrifos, chlorfenvinphos, diazinon, fenitrothion, and parathion-methyl Dimethoate, diazinon, pirimiphos methyl, parathion methyl, malathion, fenthion, chlorpyrifos, methidathion, and azinphos methyl 14 organophosphorus pesticides

Tomato, apple, carrot, and cabbage

Diazinon, parathion-methyl, parathion, malathion, and fenitrothion

Bovine liver

Olive oil

Soybean oil, peanut oil, and sesame oil Fruits: apple, pear, grapes, peaches

Diazinon, fenitrothion, fenthion, parathion ethyl, bromophos methyl, bromophos ethyl, and ethion 48 multiclass pesticides (including 12 OPS)

Cherries and strawberries

Monocrotophos, phorate, dimethoate, parathion-methyl, malathion, fenitrothion, fenthion, chlorpyrifos, parathion, methidathion, triazophos, and ethion

Cucumber and potato

202

Honey

Extraction and Cleanup Procedures Solid-phase microextraction (SPME) and stir bar sorptive extraction (SBSE) using polydimethylsiloxane as coating Liquid partitioning with acetonitrile followed by dispersive solid-phase extraction with primary secondary amine

Determination

References

LC-MS (ESI+) SIM mode

Blasco et al. 2004

LC-MS (ESI+) SIM mode

Min et al. 2005

Matrix solid phase dispersion using C18 as sorbent, silicagel for cleanup and acetonitrile as eluent Microwave assisted liquid-liquid extraction with acetonitrile: dichlormethane and cleanup with solid-phase extraction with an Envicarb cartridge

HPLC diode array detection

García de Llasera et al. 2009

GC-MS/MS and GC-FPD

Fuentes et al. 2008

Liquid partitioning with acetonitrile followed by cleanup by low temperature precipitation Solid-phase microextraction using polydimethylsiloxane (PDMS, 100 μm) of a 5-mL water fruit homogenate (5 g of fruit) Headspace solid-phase microextraction (HS-SPME) with a 100-μm polydimethylsiloxane fiber of a solution containing fruit homogenate, water, and methanol Liquid partitioning of honey (mixed with water) with ethyl acetate followed by SPE cleanup with Florisil Stir bar sorptive extraction (SBSE) with hydroxyterminated polydimethylsiloxane (PDMS)

GC-FPD

Li et al. 2007

GC-NPD

Fytianos et al. 2006

GC-MS SIM mode

Lambropoulou et al. 2003

GC-MS SIM mode

Rissato et al. 2007

GC-TSD

Wenmin et al. 2005

Table 6.2. Selected sample preparation methods for OP pesticide analysis. (cont.) Targeted Analytes

Sample/Matrix

Extraction and Cleanup Procedures

Determination

References

Ethoprophos, malathion, diazinon, parathion-methyl, isocarbophos, and quinalpos Phorate, diazinon, methyl-parathion, parathion, fenitrothion, malathion, fenthion, methidathion Diazinon, parathion-methyl, fenitrothion, malathion, chlorpyrifos, phenthoate, methidathion, profenofos, and ethion Ethoprofos, dimethoate, diazinon, malaoxon, chlorpyrifosmethyl, fenitrothion, malathion, chlorpyrifos, fenamiphos, buprofezin, and phosmet 160 multiclass pesticides

Orange juice

Single-drop microextraction using 1.6 μL of toluene as solvent

GC-FPD

Ercheng et al. 2006

Wine and fruit juices

Solid-phase microextraction using a 85 μm polyacrylate film. Juices were centrifuged before SPME

GC-MS SIM mode

Zambonin et al. 2004

Fruit juices

Matrix solid-phase dispersion with Florisil as sorbent and ethyl acetate assisted by sonication as eluting solvent

GC-NPD and GC-MS (SIM mode)

Albero et al. 2003

Bananas

QuEChERS procedure: liquid partitioning with acetonitrile followed by cleanup with primary secondary amine by dispersive solid-phase extraction

GC-NPD

HernándezBorges et al. 2009

Tomato, pear, and orange

LC-MS/MS ESI(+) MRM mode

Kmellar et al. 2008

140 multiclass pesticides

Cucumber and orange

GC-MS/MS triple quad MRM mode

FernándezMoreno et al. 2008

50 organophosphorus pesticides

Rice

QuEChERS procedure: liquid partitioning with acetonitrile followed by cleanup with primary secondary amine by dispersive solid-phase extraction QuEChERS procedure: liquid partitioning with acetonitrile followed by cleanup with primary secondary amine by dispersive solid-phase extraction Liquid partitioning with ethyl acetate followed by GPC cleanup

GC-NPD

Liu et al. 2005

GC-FPD, GC-flame photometric detector; GC-TSD, thermoionic specific detection; GC-NPD, nitrogen phosphorus detection.

203

204

Analysis of Endocrine Disrupting Compounds in Food

Acetonitrile extraction

15 mL MeCN (1% HAc) 15 g baby food

Centrifuge 3700 rpm (3min)

Shake with hands

+ 6 g MgSO4 + 1.5 g NaCl PTFE tube 50mL

Take up 5 mL of supernatant

Clean-up

Centrifuge 3700 rpm (3min)

Shake with hands

+ 0.75 g MgSO4 +0 0.25 25 g PSA PTFE tube 15mL

Take up 3 mL of supernatant

Evaporate + reconstitute

Evaporate under N2 stream

Redissolve with 0.5 mL MeOH + 1 mL H2O

Filter (0.45 µm) and transfer into a vial

Figure 6.2. Scheme of QuEChERS extraction procedure for sample treatment and extraction of multiclass pesticides from foodstuffs. It combines a liquid partitioning step with acetonitrile followed by a cleanup step with dispersive solid-phase extraction. For details, see Anastassiades et al. (2003).

(Figure 6.2). This method has been extensively applied for large-scale multiresidue methods covering dozens of OP pesticides with a wide range of polarities and has obtained satisfactory recoveries. For liquid samples, solid-phase extraction (SPE) has been applied to the isolation of OP pesticides in foodstuffs (Ridgway et al. 2007). Solid-phase microextraction (SPME) has also been proposed for OP extraction. For instance, the use of headspace SPME has been reported for the determination of OP pesticides in olive oil (Tsoutsi et al. 2006), allowing the simultaneous control of a wide range of pesticides, minimizing sample manipulation, and avoiding the use of organic solvents. The tendency toward the development of an environmentally friendly solvent-free sample treatment procedure has fostered the development of new strategies. Among them, SPME, stir bar sorptive extraction (SBSE), and matrix solid-phase dispersion (MSPD)

have been proposed for the extraction of multiclass pesticides from foodstuffs. SPME is an easy and fast technique that avoids the use of toxic solvents and can be easily automated (Beltran et al. 2000). SPME is performed by immersion of a silica fiber coated with a stationary phase in an aqueous sample and extracted by stirring the sample with a stir bar covered with poly(dimethylsiloxane) (PDMS) for a given time. The analyte enrichment is done by partitioning between the polymer and the aqueous phase, according to both their distribution factors and the desorption process, by temperature in the injector (for GC) or by liquid removal (for LC). Several works report the usefulness of these two techniques for pesticide analysis in fruits, vegetables, and other foodstuffs such as honey (Blasco et al. 2002; Blasco et al. 2003; Weinrich et al. 2001). However, it also has some drawbacks, such as relatively high cost,

Pesticides: Organophosphates

sample carryover, and a decline in performance with time. SBSE was developed and used for the determination of volatile and semivolatile organic compounds, including pesticides, in various samples (Vercauteren et al. 2001). The extraction phases of SBSE are slices of special PDMS tubing that cover a glass tube with a magnetic core. The volume and surface area of the extraction phase are 50 to 200 and 400 times more, respectively, than those of SPME. The lowest detection limit with SBSE reaches sub-microgram per liter or even nanogram per liter level. The extraction phase on the stir bar in SBSE is critical for the performance of both extraction and thermal desorption. MSPD is a simple and fast sample preparation technique for the treatment of liquid, viscous, and semisolid samples (Barker et al. 2007; Barker et al. 2000a, b). MSPD has also been used for the determination of OP pesticides in food. This technique is based on the dispersion of the sample on a sorbent such as Florisil, C18, alumina, or silica, allowing the simultaneous disruption and homogenization of solid and semisolid samples, as well as the extraction, fractionation, and cleanup of analytes in a single step. For solid samples, the mixture is done in a mortar and then transferred to the extraction columns, whereas for liquid samples, the dispersion of the matrix is done right on the extraction column. The main advantages of MSPD over classic liquid partitioning procedures of sample treatment include short extraction times, use of smaller amounts of solvents, and reduced sample handling, because this means that it is possible to simultaneously perform the extraction and (preliminary) cleanup of the analytes while still providing similar extraction efficiencies. Recently, a simple, quick, inexpensive, and virtually solvent-free sample preparation method has been developed for extraction of analytes from aqueous extracts. This technique is known as liquid-phase microextrac-

205

tion (LPME) or single-drop microextraction (SDME) (He and Lee 1997; Psillakis and Kalogerakis 2002). The technique is based on the principle that distribution of analytes must be between a microdrop of extraction solvent at the tip of a microsyringe needle and the aqueous phase. The microdrop is exposed to an aqueous sample where analyte is extracted into the drop. After extraction, the microdrop is retracted back into the microsyringe and injected into the instruments, such as GC or LC for further analysis. This technique has been applied to the determination of OP pesticides in juices (Ercheng et al. 2006).

Determination of OP pesticides Chromatographic methods for the determination of OP pesticides in food In the past decades, the methods for trace level determination of pesticides have changed considerably. Since the early 1970s, most routine pesticide residue analysis has been conducted by gas chromatography (GC) in combination with electron capture, nitrogen-phosphorus, and/or flame photometric detection. Confirmation of results required the use of a gas chromatograph equipped with a different type of column or detector, such as a mass spectrometry, which is the more widely used approach currently. Actually, according to the guidelines proposed by the Directorate General for Health and Consumer Affairs (DG SANCO) (European Commission 2006), these detectors do not provide enough selectivity to confirm the presence of a substance. In this sense, the EU has established an identification criterion for contaminants, which involves the use of MS techniques to meet the confirmation criteria that are based on the use of identification points (a different number of points is assigned according to the type of MS detection used, e.g., MS/MS, high resolution, or SIM mode) (EC 2002), although this applies only for veterinary residues so far.

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Analysis of Endocrine Disrupting Compounds in Food

Therefore, the use of chromatographic techniques coupled with mass spectrometric detection has become almost mandatory in order to obtain unambiguous identification (Lehotay et al. 2008). Today, the combined use of GC with MS provides unambiguous identification and quantitation of pesticide residues in one analytical run, avoiding matrix interference and both false positives and false negatives. Selectivity of GC-MS can also be adjusted by the selection of appropriate molecular and fragment ions to avoid interference from coextracted sample materials. In the last 15 years, GC-MS was the technique of choice for pesticide analysis in food. However, the tendency toward the use of a large number of newly authorized pesticides and commercial formulations in crops which conform to integrated pest-management guidelines, and the fact that these new substances are more polar, has prompted the use of LC-MS as a complementary tool to GC-MS, particularly for compounds not amenable to, or with a poor performance by GC. The commercial availability of liquid chromatography mass spectrometry (LCMS) instruments of atmospheric pressure ionization has caused a dramatic change in the last few years. Compared to traditional detectors, electrospray (ESI) and atmospheric pressure chemical ionization (APCI), in combination with MS instruments, have increased the sensitivity of LC detection by several orders of magnitude, particularly if tandem mass spectrometers are used and operated in the multiple reaction monitoring (MRM) mode. In a recent study from Alder and coworkers (2006), for a total of 500 pesticides (selected as the most important in Germany) LC-MS gave better results than GC-MS. Of the 500 compounds tested, 453 were amenable to LC-MS, and only 365 could be analyzed by GC-MS. In the case of OP compounds (80 in the cited study), most of the compounds (see Table 6.3) showed better sensi-

tivity by LC-MS. Note that the data are taken from a single study, but in general terms, most organophosphorus pesticides are relatively polar, particularly those with small molecular weight or the main transformation products. In these cases, the sensitivity of GC-MS is poor. Only in the case of nonpolar OP pesticides, that is, those that appear at the end of a reversed-phase LC run (with an organic solvent gradient up to 100% of organic mobile phase), does the sensitivity by GC-MS compare well with that attainable by LC-MS methodologies. Therefore, it is clear LC-MS becomes the best option for a wide-range method covering all the OP pesticides, as shown in Table 6.3. In contrast to GC-MS, single quadrupole mass spectrometers are not used in the majority of recent studies dealing with LC-MS. A disadvantage of single quadrupole instruments is the high intensity of background noise signals produced from sample matrix and LC–mobile phase clusters. Due to this chemical noise in real samples, very low limits of quantification cannot be achieved, even if the sensitivity of these instruments is high (Picó et al. 2004). The chemical is reduced significantly if tandem MS in combination with multiple-reaction monitoring (MRM) mode is applied. Even if a coextracted matrix component has the molecular mass of a pesticide, usually both isobaric ions can be separated in MRM experiments because their fragmentation in the collision cell most often results in different product ions. Therefore, tandem mass spectrometers offer excellent sensitivity and unsurpassed selectivity. For this reason, triple quadrupole mass analyzers have been the most frequently applied MS detectors so far. Two MS/MS transitions are usually employed for the identification and confirmation of a targeted substance as shown in Table 6.3 for the OP pesticides included. As an alternative to LC-MS/MS, liquid chromatography coupled with time-of-flight mass spectrometers (LC-TOFMS) has also

Table 6.3. Main mass spectral features and analytical performance (LOQ) of gas chromatography/ mass spectrometry (GC-MS) and liquid chromatography/mass spectrometry (LC-MS) determination of organophosphorus pesticides in food. Pesticide

LC-MS Elemental Composition

GC-MS

Accurate Mass of Ions [M+H]+

LC-MS/MS Transitions (m/z → m/z) 184 → 143 184 → 125 325 → 183 325 → 139 318 → 132 318 → 160 346 → 132 346 → 160 395 → 339 395 → 367 271 → 159 271 → 97 359 → 155 359 → 99 350 → 97 350 → 198

Acephate

C4H10NO3PS

184.0192

Azamethiphos

C9H10ClN2O5PS

324.9809

Azinphosmethyl Azinphos-ethyl

C10H12N3O3PS2

339.9949

C12H16N3O3PS2

346.0444

Bromophosethyl Cadusafos

C10H12BrCl2O3PS

394.8854

C10H23O2PS2

271.0949

Chlorfenvinphos

C12H14Cl3O4

358.9768

Chlorpyrifos

C9H11Cl3NO3PS

349.9335

Chlorpyrifosmethyl

C7H7Cl3NO3PS

321.9022

322 → 125 322 → 290

Chlorthiophos

C11H15Cl2O3PS2

360.9650

361 → 305 361 → 333

Coumaphos

C14H16ClO5PS

363.0217

363 → 227 363 → 307

Cyanofenphos

C15H14NO2PS

304.0555

304 → 157 304 → 276

Cyanophos

C9H10NO3PS

Diazinon

C12H21N2O3PS

244.0191 261.0457* 305.1083

Dichlorvos

C4H7Cl2O4P

Dicrotophos

C8H16NO5P

LOQ* LC-MS2 (ng/mL)

Fragment Ions in GC-EI-MS (m/z)

References LOQ GC-MSa (ng/mL)

0.1

142;136;125

100

0.1

215;155;125

10,000

0.1

160;132;77

100

0.1

160;132;104

1000

10

359;357;303

1

1

270;214;159

10

0.1

323;269;267

10

1

316;314;286

10

10

288;286;197

1

0.1

360;325;269

100

0.1

362;226;210

100

10

185;169;157

1

261 → 125 261 → 212 305 → 169 305 → 97

100

243;180;125

100

0.1

304;179;152

10

220.9532

221 → 109 221 → 127

1

185;149;109

100

238.0839

238 → 112 238 → 127

0.1

237;193;127

100

Fillion et al. 2000 Pang et al. 2006 Fillion et al. 2000 Fillion et al. 2000 Fillion et al. 2000 Pang et al. 2006 Fillion et al. 2000 Fillion et al. 2000; Hernández et al. 2001 Hayward et al. 2009; Hetherton et al. 2004 Alder et al. 2006; Fillion et al. 2000; Pang et al. 2006 Alder et al. 2006; Fillion et al. 2000; Pang et al. 2006 Alder et al. 2006; Fillion et al. 2000; Pang et al. 2006 Fillion et al. 2000 Fillion et al. 2000; GarcíaReyes et al. 2007b Fillion et al. 2000; Hetherton et al. 2004 Fillion et al. 2000; Lehotay et al. 2005

(continued)

207

Table 6.3. Main mass spectral features and analytical performance (LOQ) of gas chromatography/ mass spectrometry (GC-MS) and liquid chromatography/mass spectrometry (LC-MS) determination of organophosphorus pesticides in food. (cont.) Pesticide

LC-MS Elemental Composition

GC-MS

Accurate Mass of Ions [M+H]+

LC-MS/MS Transitions (m/z → m/z)

LOQ* LC-MS2 (ng/mL)

Fragment Ions in GC-EI-MS (m/z)

References LOQ GC-MSa (ng/mL)

Dimethoate

C5H12NO3PS2

230.0075

230 → 199 230 → 125

0.1

229;143;125

100

Edifenphos

C14H15O2PS2

310;173;110

100

C9H22O4P2S4

0.1

384;231;203

10

Ethoprophos

C8H19O2PS2

243.0636

328 → 109 328 → 283 385 → 199 385 → 171 243 → 131 243 → 97

1

Ethion

311.0323 328.0589* 384.9949

0.1

200;158;139

10

Fenamiphos

C13H22NO3PS

304.1131

304 → 217 304 → 202

0.1

303;260;217

1000

Fenitrothion

C9H12NO5PS

278.0247

278 → 125 278 → 109

277;260;125

100

Fenthion

C10H15O3PS2

279.0273

279 → 169 279 → 247

0.1

278;245;169

10

Fonofos

C10H15OPS2

247.0375

247 → 109 247 → 137

1.0

246;170;136

Isofenphos

C15H24NO4PS

346.1236

0.1

255;213;185

Isofenphos methyl Malaoxon

C14H22NO4PS

332.1079

0.1

121;199;241;231

C10H19O7PS

315.0662

346 → 217 346 → 245 332 → 221 332 → 121 315 → 127 315 → 99

0.1

268;195;173

1000

Malathion

C10H19O6PS2

331.0433

331 → 127 331 → 99

0.1

285;256;173

100

Methamidophos

C2H8NO2PS

142.0086

0.1

141;126;111

1000

Methidathion

C6H11N2O4PS3

324.9511

142 → 94 142 → 125 303 → 145 303 → 85

0.1

146;145;125

100

Methomyl

C5H10N2O2S

185.0355

1

105;88;73

100

Mevinphos

C7H13O6P

225.0523

0.1

192;164;141

100

Monocrotophos

C7H14NO5P

224.0682

163 → 88 163 → 106 225 → 127 225 → 193 224 → 127 224 → 98

1

192;164;127

1000

Omethoate

C5H12NO4PS

214.0297

214 → 125 214 → 109

0.1

262;156;110

1000

100

1.0

10 5

Fillion et al. 2000; Hayward et al. 2009 Pang et al. 2006 Pang et al. 2006 Fillion et al. 2000; Pang et al. 2006 Hetherton et al. 2004; Lehotay et al. 2005 Pang et al. 2006; Bossi et al. 2002 Fillion et al. 2000; Hetherton et al. 2004 Fillion et al. 2000; Hetherton et al. 2004 Mezcua et al. 2009a Mezcua et al. 2009a García-Reyes et al. 2007b Pang et al. 2006; GarcíaReyes et al. 2007b Klein et al. 2003 Fillion et al. 2000; Hayward et al. 2009 Klein et al. 2003 Fillion et al. 2000 Fillion et al. 2000; Klein et al. 2003 Fillion et al. 2000; Klein et al. 2003

(continued)

208

Pesticides: Organophosphates

209

Table 6.3. Main mass spectral features and analytical performance (LOQ) of gas chromatography/ mass spectrometry (GC-MS) and liquid chromatography/mass spectrometry (LC-MS) determination of organophosphorus pesticides in food. (cont.) Pesticide

LC-MS Elemental Composition

a

GC-MS

Accurate Mass of Ions [M+H]+

LC-MS/MS Transitions (m/z → m/z)

LOQ* LC-MS2 (ng/mL)

Fragment Ions in GC-EI-MS (m/z)

References LOQ GC-MSa (ng/mL)

Paraoxon

C10H14NO6P

276.0631

276 → 220 276 → 94

0.1

275;149;139

1000

Paraoxonmethyl

C8H10NO6P

248.0318

248 → 202 248 → 109

0.1

247;230;200

1000

Parathion

C10H14NO5PS

292.0403

292 → 236 292 → 97

10

291;235;218

100

Parathion methyl

C8H10NO5PS

263.0011

264 → 125 264 → 232

10

263;233;125

100

Phosmet

C11H12NO4PS2

318.0018

318 → 160 318 → 133

1.0

317;286;161

100

Pirimiphos-ethyl

C13H24N3O3PS

334.1348

334 → 198 334 → 182

0.1

333;318;304

10

Pirimiphosmethyl

C11H20N3O3PS

306.1036

306 → 164 306 → 108

0.1

305;290;276

10

Propetamphos

C10H20NO4PS

282.0923

0.1

236;194;156

100

Terbufos

C9H21O2PS3

289.0514

282 → 138 282 → 156 289 → 103 289 → 57

0.1

288;231;203

10

Fillion et al. 2000; Mezcua et al. 2009b Pang et al. 2006; Mezcua et al. 2009b Pang et al. 2006; Mezcua et al. 2009b Pang et al. 2006; Mezcua et al. 2009b Fillion et al. 2000; Pang et al. 2006 Fillion et al. 2000; Hayward et al. 2009 Fillion et al. 2000; Sancho et al. 2004 Mezcua et al. 2009b Fillion et al. 2000; Hetherton et al. 2004

LOQ data adapted from Alder et al. (2006).

been proposed recently for the analysis of organophosphorus pesticides in food (Mezcua et al. 2008). As an example, the identification of isofenphos-methyl, an EU-banned insecticide, detected in green peppers is shown in Figure 6.3. The identification in this case is based on the combination of retention time and accurate mass measurements of the characteristic ions of the compound targeted (protonated molecule and in-source CID fragment ions). The main advantage of this type of instrument is the identification of unknown

compounds that can be used to identify transformation products of organophosphorus pesticides in food (García-Reyes et al. 2007a). However, this advantage is usually not required in the enforcement of maximum residue levels. Table 6.3 contains the main information regarding the mass spectral features of 40 relevant OP pesticides. Typical ions selected for GC-MS analysis in SIM mode, MS/MS transitions used for LC-MS/ MS analysis in MRM mode, and accurate masses of ions used for LC-TOFMS analysis

(a)

6.0e7

y, cps y, Intensity

5.2e7 4.4e7 3.6e7 2.8e7 28 7 2.0e7 1.2e7 4.0e6 2

4

6

8

10

12

14 12.30

ensity, cps ensity, Inte

7.0e4

16 18 Time, min

20

22

24

26

28

30

32

34

(b)

6.0e4 5 0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0 14.0 Time, min

15.0

193.0862

3.8e4 3.4e4 3.0e4 2.6e4 2.2e4 1.8e4 1.4e4 1.0e4 6000.0

16.0

17.0

18.0

19.0

20.0

21.0

22.0

(c)

209.1535

Inttensity, tensity, counts

271.0955 227.1674 131.0490

161.0598

273.0918

163.0800 103.0530 114.0926

80

100

191.1432

133.0769

120

140

160

180 m/z, amu

211.0977

200

251.1635

225.0999 220

240

260

280

(d)

27.83

8.0e5 Intensity, cps

7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 23.0

24.0

25.0

26.0

27.0

28.0 Time, min 230.9879

29.0

30.0

31.0

32.0

33.0

(e)

Intensity, counts

3.2e5 2.8e5 2.4e5 2.0e5 1.6e5 1.2e5

[M+Na]+ [M+H]+ 354.0903 332.1072

273.0348

8.0e4 4.0e4 60

80

100

120

140

160

180

200

220 240 m/z, amu

260

280

300

320

340

360

380

Figure 6.3. (a) Total ion chromatogram corresponding to the LC-TOFMS analysis of a pepper sample (#38655), where nitenpyram (0.11 mg kg−1) and isofenphos-methyl (0.07 mg kg−1) were detected. (b) Extracted ion chromatogram of nitenpyram. (c) Accurate mass spectrum obtained at 12.30 minutes. (d) Extracted ion chromatogram of isofenphos-methyl. (e) Accurate mass spectrum obtained at 27.82 minutes. Reproduced from Mezcua et al. (2009a), with permission.

210

Pesticides: Organophosphates

of selected organophosphorus pesticides are included.

Nonchromatographic (fast) methods for the screening and determination of OP pesticides in food Besides chromatographic methods, there is a vast array of analytical techniques that have been proposed for the detection of OP pesticides in foodstuffs. These techniques are expected to be specific, sensitive, cost-effective, rapid, and suitable for field analysis, while skipping the complexity and time required for a thorough chromatographic analysis. This section intends to provide insight into the different approaches proposed for the development of fast screening methods for OP pesticides in food. We have focused our attention on the techniques that have been explored by several authors. Among them, enzyme-linked immunoassays (ELISAs), biosensors, chemiluminescencebased assays, and ambient mass spectrometry have been used successfully in the development of effective methods for OP detection in food. Biological methods such as immunoanalytical techniques (i.e., ELISA) and enzymebased electrochemical biosensors are probably among the more popular techniques for the rapid detection of organophosphorus pesticides. However, we should emphasize that sample treatment is still required when food samples are analyzed. Therefore, the advantages of the methods presented here related to the dedicated chromatographic methods rely on the speed and throughput of the determination step, together with the field-analysis capabilities and the reduced cost of the instrumentation required. ELISA Immunoassays emerged as an alternative to the traditional chromatographic methods in the 1990s. Immunochemical techniques that

211

have been used extensively in clinical laboratories recently began to gain acceptance as fast, sensitive, and cost-effective tools for detecting trace amounts of pesticides (Nunes et al. 1998). The enzyme-linked immunosorbent assay (ELISA), known as a simple, rapid, and cost-effective method for monitoring pesticide residue, and especially known for its capacity for high-throughput screening analysis, has been demonstrated as an alternative to traditional instrument analysis methods (Hennion and Barcelo 1998; Marco et al. 1995). The development of an immunoassay requires the production of antibodies to the analyte. Since pesticides are small molecules, pesticide derivatives, namely haptens, must be synthesized and coupled to carrier proteins to induce antibody production. One type of hapten for organophosphorus pesticides has an aminocarboxylic acid bridge at the thiophosphate group and has been used successfully in the development of ELISA for several OP pesticides (Cho et al. 2004). The ELISAs are usually performed in laboratories using microtiter plates and, thus, are not suitable for field tests. As an example of the ELISA method for pesticide detection in foodstuff, GarcésGarcía et al. (2006) described a method based on ELISA for the determination of four organophosphorus pesticides in olive oil. The analytical procedure involved simultaneous extraction of the analytes from the oil matrix with methanol and a low-temperature cleanup, followed by immunoassay determination using matrix-matched standards. The methodology was specific for diazinon, fenthion, malathion, and chlorpyrifos, showing little or no cross-reactivity against other organophosphorus compounds. Limits of detection for the pesticides in olive oil were from 46 μg/L down to 10 μg/L for fenthion. In order to extend the application of ELISA to a field test, dipstick immunoassays were proposed for the control of organophosphorus pesticides in food (Cho et al. 2003).

212

Analysis of Endocrine Disrupting Compounds in Food

This method allows a qualitative on-site determination of analytes. Dipstick immunoassays usually follow the standard ELISA procedure but use a membrane as antibodycoating support and rely on color development as seen with the naked eye. Using this approach, Cho et al. (2003) used antibodies and an enzyme tracer to develop a competitive antibody-coated (direct) ELISA for the insecticide fenthion in both microtiter plate and dipstick formats. The microtiter plate ELISA showed a IC50 value of 1.2 μg/L, with a detection limit of 0.1 μg/L. A similar approach was developed by the same authors for the detection of bromophos-ethyl (Kim et al. 2002) and isofenphos (Lee et al. 2006) in crop samples, with detection limits of 1.0 μg/L and 4.8 μg/L, respectively. The use of the dipstick format using Immunodyne as a support membrane allowed the quick visual detection of fenthion at concentrations higher than 10 μg/L. The dipstick format ELISA method detection limit using reflectance detection was 0.5 μg/L. One of the main weaknesses of these methods is that they only target a single pesticide. Therefore, the number of assays required in order to monitor the more relevant species is high, considering that today chromatographic methods cover over 100 species in a single run that takes no longer than 20 minutes using state-ofthe-art methods (Fernández-Alba et al. 2008). The demand for cost-effective fast screening of multipesticide residues has prompted the development of class-specific immunoassays. In this sense, Liang et al. (2008) have recently proposed a broad classspecific ELISA for the screening of O,Odimethyl organophosphorus pesticides, including malathion, dimethoate, phenthoate, phosmet, methidathion, fenitrothion, methyl parathion, and fenthion. The use of such a general assay has several advantages, such as screening out samples containing low-level residues in order to avoid further instrumental analysis.

Biosensors A biosensor is an analytical tool that may be defined as a combination of a biological recognition element (for example enzyme, whole cell, antibody, microbe) and a physical transducer in intimate contact with each other. The role of the transducer is to relate the concentration of an analyte into a chemical and/or physical property, which is sequentially sensed, converted into an electrical/optical signal, and amplified. Many transduction principles are available, including photometry, fluorimetry and chemiluminescence, fiber optics, piezoelectric sensing, and potentiometric and amperometric electrodes. Despite remarkable improvements in the enzyme immobilization techniques for electrode construction that have substantially contributed to detection of several contaminants at trace levels, the application of the electrochemical biosensors for the determination of pesticides in foodstuffs is limited. Analytical methods employing enzyme sensors based on the inhibition of cholinesterase with potentiometric or amperometric detection have been developed for the determination of OP pesticide insecticides (Jaffrezic-Renault 2001). Despite the high sensitivity and reproducibility of some ChEbased sensors, the development of methods employing biosensors to pesticide-residue monitoring in food samples is limited. Biosensors are sensitive and can be used as disposable sensors. These biosensors are based on inhibition of acetylcholinesterases by organophosphorus compounds or on inhibition of enzyme phosphatases (acid or alkaline) or on direct detection of organophosphorus compounds by organophosphorus hydrolase. Biosensing analytical devices, based on the acetylcholinesterase (AChE) inhibition test, using AChE-modified amperometric transducers (measuring thiocholine and p-aminophenol produced by hydrolysis of butyrylthiocholine and p-aminophenyl

Pesticides: Organophosphates

acetate, respectively, or hydrogen peroxide generated as a result of the oxidation of choline produced from acetylcholine hydrolysis in the presence of choline oxidase) have been reported (Periasamy et al. 2009). Potentiometric transducers (measuring the pH change as a result of acetic acid production) have also been reported. Biosensors based on AChE inhibition have some limitations. First, AChE is inhibited by neurotoxins, including not only OP pesticides but also carbamate and many other compounds, and these analytical tools are not selective and cannot be used for quantitation of either an individual or a class of pesticides (Mulchandani et al. 2001). Second, these protocols involve multiple steps, requiring measurement of the uninhibited activity of AChE, followed by incubation of the sensor with the analyte sample for 10–15 min (or even longer) and the measurement of the AChE again to determine the degree of inhibition. A final step or reaction/regeneration (which in many cases is partial and in some cases not possible due to irreversible inhibition) is necessary if the electrode is to be reused. As an alternative organophosphorus hydrolase (OPH) is an organophosphotriesterhydrolyzing enzyme first discovered in the soil microorganisms Pseudomonas diminuta. The enzyme has broad substrate specificity and is able to hydrolyze a number of OP pesticides such as paraoxon, parathion, coumaphos, diazinon, and methyl-parathion (Schöning et al. 2003). OPH-catalyzed hydrolysis of OP compounds generates two protons as a result of the cleavage of the P-O, P-F, P-S or P-CN bonds and an alcohol, which in many cases is chromophoric and/or electroactive. This enzyme reaction can be combined with a variety of transduction schemes to construct simple biosensors for the direct and rapid determination of OPs: potentiometric transducers (pH electrodes), pH indicator dyes, optical transducers to monitor p-nitrophenol, and amperometric transducers to monitor the

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oxidation or reduction current of the hydrolysis products. Chemiluminescence assays A cheap alternative to immunoassays, biosensors, and chromatographic methods is the use of chemiluminescence detection. Chemiluminescence, particularly when combined with flow injection analysis (FIA) systems is an attractive analytical method offering sensitive and rapid detection, with simple handling for on-line or real-time monitoring. For instance, Li et al. (2008) developed a chemiluminescence method for the quantitative assay of the organophosphorus pesticide chlorpyrifos in vegetable samples based on the enhancing effect of chlorpyrifos on the luminol/H2O2 reaction in alkaline media. The method was successfully applied to the determination of chlorpyrifos residues in vegetable samples. The method was able to detect as low as 3.5 μg/L of the targeted compound. Other similar methods have been proposed for organophosphorus pesticides (Moris et al. 1995; Roda et al. 1994; Wang et al. 2001). The main drawback of these methods is the poor selectivity for the targeted compound. There are several potential interfering species that may affect the CL signal. A similar approach was proposed by Song et al. (2002) using a FIA system with the CL reagents (luminol and periodate) immobilized using anion-exchange columns as shown in Figure 6.4. Increasing concentrations of chlorpyrifos gave rise to a decrease in the CL signal of the luminol/periodate CL reaction. The proposed approach with a limit of detection of 0.48 ng/mL was applied to detect chlorpyrifos residues in oranges. Ambient mass spectrometry As an alternative to gas chromatographybased or liquid chromatography-based methods with selective and/or mass spectrometric detection, a new family of mass

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(a)

Pump NaOH Eluant

Flow Cell Detector

Anion Exchange Column Mixing Tubing

Carrier Valve

Sample

Waste Recorder

500

I

(b)

Relative CL intensity

400

II

300 III 200 IV

100

0

0

0.5

1 Time (min)

1.5

2

Figure 6.4. (a) Schematic diagram of a flow injection system for chlorpyrifos determination in fruits. (b) Chemiluminescence time profile in the batch system: (I) CL intensity in the absence of chlorpyrifos; (II) CL intensity in the presence of 2.5 ng/mL of chlorpyrifos; (III) CL intensity in the presence of 25 ng/mL of chlorpyrifos; (IV) CL intensity in the presence of 250 ng/mL of chlorpyrifos. Reproduced from Song et al. (2002), with permission.

spectrometric techniques has emerged that allows ions to be created from condensed phase samples under ambient conditions and then collected and analyzed by mass spectrometry. This innovation in mass spectrometry, called ambient ionization mass spectrometry (Cooks et al. 2006; Venter et al. 2008), allows the acquisition of mass spectra on ordinary solid or liquid samples in their native environment, without sample preparation or removal of the matrix by creating ions from surfaces outside the instrument. This feature is unique because all the other methods described usually require stages of sample preparation (homogenization, extraction, cleanup). These techniques enable even nondestructive/invasive detection of pesticides on the peels of fruits and vegetables. It

was recently proposed that this approach use desorption electrospray ionization mass spectrometry (DESI-MS) for the detection of multiclass pesticides, including malathion and isofenphos-methyl in fruit and vegetable peels (García-Reyes et al. 2009). Another ambient ionization technique, called atmospheric pressure glow discharge mass spectrometry (APGD-MS) method, was also proposed for the screening of pesticides on fruit skins and in fruit juices (Jecklin et al. 2008). Direct analysis of market samples without any further treatment was demonstrated in both techniques. Future work in this field includes the use of DESI-MS, APGDMS, and other similar techniques for fieldoriented pesticide testing, using handheld mass spectrometers (Ouyang et al. 2009).

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References Albero, Beatriz; Sánchez-Brunete, Consuelo; Tadeo, Jose Luis. 2003. Determination of organophosphorus pesticides in fruit juices by matrix solid-phase dispersion and gas chromatography. Journal of Agricultural and Food Chemistry 51(24):6915–6921. Alder, Lutz; Greulich, Kerstin; Kempe, Günther; Vieth Bärbel. 2006. Residue analysis of 500 high priority pesticides: Better by GC-MS or LC-MS/MS? Mass Spectrometry Reviews 25(6):838–865. Anastassiades, Michelangelo; Lehotay, Steven J.; Stajnbaher, Darinka; Schenk, Frank J. 2003. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. Journal of AOAC International 86(2): 412–431. Andersen, H.R.; Cook, S.J.; Waldbillig, D. 2002. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicology and Applied Pharmacology 197(1):1–12. Barker, Steven A. 2007. Matrix solid phase dispersion (MSPD). Journal of Biochemical and Biophysical Methods 70(2):151–162. Barker, Steven A. 2000a. Applications of matrix solidphase dispersion in food analysis. Journal of Chromatography A 880(1–2):63–68. Barker, Steven A. 2000b. Matrix solid phase dispersion. Journal of Chromatography A 885(1–2):115–127. Beltrán, Joaquin; López, Francisco J.; Hernández, Félix. 2000. Solid-phase microextraction in pesticide residue analysis. Journal of Chromatography A 885(1–2): 389–404. Blasco, Cristina; Font, Guillermina; Picó, Yolanda. 2002. Comparison of microextraction procedures to determine pesticides in oranges by liquid chromatography-mass spectrometry. Journal of Chromatography A 970(1–2):201–210. Blasco, Cristina; Font, Guillermina; Mañes, Jordi; Picó, Yolanda. 2003. Solid-phase microextraction liquid chromatography/tandem mass spectrometry to determine postharvest fungicides in fruits. Analytical Chemistry 75(14):3606–3615. Blasco, Cristina; Fernández, Mónica; Picó, Yolanda; Font, Guillermina. 2004. Comparison of solid-phase microextraction and stir bar sorptive extraction for determining six organophosphorus insecticides in honey by liquid chromatography-mass spectrometry. Journal of Chromatography A 1030(1):77–85. Bossi, Rossana; Vejrup, Karl V.; Mogensen, Betty Bügel; Asman, Willem A.H. 2002. Analysis of polar pesticides in rainwater in Denmark by liquid chromatography tandem mass spectrometry. Journal of Chromatography A 957(1):27–36. Boujelben, Ghazi; Jamoussi, Kamel; Ammar, Emna; Ellouze, Fériel; Guermazi, Fadhel; Zeghal, Najiba. 2005. Effects of dimethoate on bone maturation of young rats during the suckling period. Pesticide Biochemistry and Physiology 83(2–3):132–139. Bustos, O.E. 2001. Parathion induces mouse germ cells apoptosis. Italian Journal of Anatomy and Embryology 106(2):199–204.

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Cabello, G.; Valenzuela, M.; Vilaxa, A.; Durán, V.; Rudolph, I.; Hrepic, N.; Calaf, A. 2001. A rat mammary tumor model induced by the organophosphorus pesticides parathion and malathion, possibly through acetylcolinesterase inhibition. Environmental Health Perspectives 109(5):471–479. Cabello, G.; Jurranz, A.; Botella, L.M.; Calaf, G.M. 2003. Organophosphorus pesticides in breast cancer progression. Journal of Submicroscopic Cytology and Pathology 35(1):1–9. Cho, Young Ae; Kim, Yoo Jung; Hammock, Bruce D.; Lee, Yong Tae; Lee, Hyr-Sung. 2003. Development of a microtiter plate ELISA and a dipstick ELISA for the determination of the organophosphorus insecticide fenthion. Journal of Agricultural and Food Chemistry 51(27):7854–7860. Cho, Young Ae; Seok, J.-A; Lee, Hye-Jin; Lee, HyeSung; Kim, Yoo Hung; Park, Yong Chae; Lee, Yong Tae. 2004. Synthesis of haptens of organophosphorus pesticides and development of immunoassays for fenitrothion. Analytica Chimica Acta 522(2):215–222. Cocco, P. 2002. On the rumours about the silent spring. Review of the scientific evidence linking occupational and environmental pesticide exposure to endocrine disruption health effects. Cadernos de Saude Publica 18(2):379–402. Cooks, R. Graham; Ouyang, Zheng; Takats, Zoltan; Wiseman, Justin M. 2006. Detection technologies. Ambient mass spectrometry. Science 311(5767): 1566–1570. Ercheng, Zhao; Lijun, Han; Shuren, Jiang; Qiuxia, Wang; Zhiqiang, Zhou. 2006. Application of a singledrop microextraction for the analysis of organophosphorus pesticides in juice. Journal of Chromatography A 1114(1–2):269–273. European Commission (EC). 2002. Decision of 12 August 2002, implementing Council Directive 96/23/ EC concerning the performance of analytical methods and the interpretation of results. Official Journal European Union 221:8. European Commission (EC). 2006. Quality Control Procedures for Pesticide Residues Analysis, DG SANCO/10232/2006, 24 March 2006, and subsequent updates. Available at: http://ec.europa.eu/food/plant/ protection/resources/qualcontrol_en.pdf Fernández-Alba, Amadeo R.; García-Reyes, Juan F. 2008. Large-scale multi-residue methods for pesticides and their degradation products in food by advances LC-MS. Trends in Analytical Chemistry 27(11):973–990. Fernández, Moreno, Jose, Luis; Garrido Frenich, Antonia; Plaza Bolaños, Patricia; Martínez Vidal, Jose Luis. 2008. Multiresidue method for the analysis of more than 140 pesticide residues in fruits and vegetables by gas chromatography coupled to triple quadrupole mass spectrometry. Journal of Mass Spectrometry 43(9):1235–1254. Fillon, Julie; Sauve, Francois; Selwyn, Jennifer. 2000. Multiresidue method for the determination of residues of 251 pesticides in fruits and vegetables by gas chromatography/mass spectrometry and liquid chromatography with fluorescence detection. Journal of AOAC International 83(3):698–713.

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Fuentes, Edwar; Baez, María E.; Quinones, Adali. 2008. Suitability of microwave-assisted extraction coupled with solid-phase extraction for organophosphorus pesticide determination in olive oil. Journal of Chromatography A 1207(1):38–45. Fytianos, K.; Raikos, N.; Theodoridis, G.; Velinova, Z.; Tsoukali, H. 2006. Solid phase microextraction applied to the analysis of organophosphorus insecticides in fruits. Chemosphere 65(11):2090–2095. Garcés-García, Marta; Brun, Eva M.; Puchades, R.; Maquieira, A. 2006. Immunochemical determination of four organophophorus insecticide residues in olive oil using a rapid extraction process. Analytica Chimica Acta 556(2):347–354. García de Llasera, Martha P.; Reyes-Reyes, Maria L. 2009. A validated matrix solid-phase dispersion method for the extraction of organophosphorus pesticides from bovine samples. Food Chemistry 114(4): 1510–1516. García-Reyes, Juan F.; Hernando, M. Dolores; Ferrer, Carmen; Molina-Díaz, Antonio; Fernández-Alba, Amadeo R. 2007b. Large scale pesticide multiresidue methods in food combining liquid chromatography— time-of-flight mass spectrometry and tandem mass spectrometry. Analytical Chemistry 79(19): 7308–7323. García-Reyes, Juan F.; Jackson, Ayanna U.; MolinaDíaz, Antonio; Cooks, R. Graham. 2009. Desorption electrospray ionization mass spectrometry for trace analysis of agrochemicals in food. Analytical Chemistry 81(2):820–829. García-Reyes, Juan F.; Molina-Díaz, Antonio; Fernández-Alba, Amadeo R. 2007a. Identification of pesticide transformation products in food by liquid chromatography/time-of-flight mass spectrometry via “fragmentation-degradation” relationships. Analytical Chemistry 79(1):307–321. Hagan, Nathan A.; Cornish, Timothy J.; Pilato, Robert S.; Van Houten, Kelly A.; Antoine, Miquel D.; Lippa, Timothy P.; Becknell, Alan F.; Demirev, Plamen A. 2008. Detection and identification of immobilized low-volatility organophosphates by desorption ionization mass spectrometry. International Journal of Mass Spectrometry 278(2–3):158–165. Hayward, Douglas G.; Wong, Jong W. 2009. Organohalogen and organophosphorus pesticide method for ginseng root–a comparison of gas chromatography single quadrupole mass spectrometry with high resolution time-of-flight mass spectrometry. Analytical Chemistry 81(14):5716–5723. He, Y.; Lee, Hian Kee. 1997. Liquid-phase microextraction in a single drop of organic solvent by using a conventional microsyringe. Analytical Chemistry 69(22):4634–4640. Hennion, Marie-Claire; Barceló, Damia. 1998. Strengths and limitations of immunoassays for effective and efficient use for pesticide analysis in water samples: A review. Analytica Chimica Acta 362(1):3–34. Hernández, F.; Sancho, J.V.; Pozo O.; Lara, A.; Pitarch, E. 2001. Rapid direct determination of pesticides and metabolites in environmental water samples at submicrog/l level by on-line solid-phase extraction-liquid

chromatography-electrospray tandem mass spectrometry. Journal of Chromatography A 939(1):1–11. Hernández-Borges, Javier; Cabrera Cabrera, Juan; Rodríguez-Delgado, Miguel Ángel; HernándezSuárez, Estrella M; Galán Sauco, Victor. 2009. Analysis of pesticide residues in bananas harvested in the Canary Islands (Spain). Food Chemistry 113(1): 313–319. Hetherton, Christel L.; Sykes, Mark D.; Fussell, Richard J.; Goodall, David M. 2004. A multi-residue screening method for the determination of 73 pesticides and metabolites in fruit and vegetables using highperformance liquid chromatography/tandem mass spectrometry. Rapid Communications in Mass Spectrometry 18(20):2443–2450. Jaffrezic-Renault, Nicole. 2001. New trends in biosensors for organophosphorus pesticides. Sensors 1(2):60–74. Jecklin, Matthias Conradin; Gámez, Gerardo; Touboul, David; Zenobi, Renato. 2008. Atmospheric pressure glow discharge desorption mass spectrometry for rapid screening of pesticides in food. Rapid Communications in Mass Spectrometry 22(18): 2791–2798. Kang, H.G.; Jeong, S.H.; Cho, J.H.; Kim, D.G.; Park, J.M.; Cho, M.H. 2004. Chlorpyrifos-methyl shows anti-androgenic activity without estrogenic activity in rats. Toxicology 199(2–3):219–230. Kim, Kwang-Ok; Kim, Yoo Jung; Lee, Yong Tae; Hammock, Bruce D.; Lee, Hye-Sung. 2002. Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl. Journal of Agricultural and Food Chemistry 50(23):6675–6682. Kitamura, Shigeyuki; Sugihara, Kazumi; Fujimoto, Nariaki. 2006. Endocrine disruption by organophosphate and carbamate pesticides. In: Toxicology of Organophosphate and Carbamate Compounds, Ramesh, Gupta, ed., Amsterdam: Elsevier, pp. 481–494. Klein, Jeannette; Alder, Lutz. 2003. Applicability of gradient liquid chromatography with tandem mass spectrometry to the simultaneous screening for about 100 pesticides in crops. Journal of AOAC International 86(5):1015–1037. Kmellar, Bella; Fodor, Peter; Pareja, Lucia; Ferrer, Carmen; Martínez-Uroz, M. Ángeles; Valverde, Antonio; Fernández-Alba, A.R. 2008. Validation and uncertainty study of a comprehensive list of 160 pesticide residues in multi-class vegetables by liquid chromatography tandem mass spectrometry. Journal of Chromatography A 1215(1):37–50. Lambropoulou, Dimitra; Albanis, Triantafyllos A. 2003. Headspace solid-phase microextraction in combination with gas chromatography-mass spectrometry for the rapid screening of organophosphorus insecticide residues in strawberries and cherries. Journal of Chromatography A 993(1–2):197–203. Lee, Won Young; Lee, Eun Kyung; Kim, Yoo Jung; Park, Won Chul; Chung, Taeowan; Lee, Yong Tae. 2006. Monoclonal antibody-based enzyme linked immunosorbent assays for the detection of organo-

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phosphorus insecticide isofenphos. Analytica Chimica Acta 557(1–2):169–178. Lehotay, Steven J.; De Kok, André; Hiemstra, Maurice; van Bodegraven, Peter. 2005. Validation of a fast and easy method for the determination of residues from 229 pesticides in fruits and vegetables using gas and liquid chromatography and mass spectrometric detection. Journal of AOAC International 88(2): 595–614. Lehotay, Steven J.; Mastovska, Katerina; Amirav, Aviv; Flatkov, Alexander B.; Martos, Perry A.; De Kok, André; Fernández-Alba, A.R. 2008. Identification and confirmation of chemical residues in food by chromatography mass spectrometry and other techniques. Trends in Analytical Chemistry 27(11):1070–1090. Li, Aifang; Liu, Xiaoyu; Kong, Juan; Huang, Rong; Wu, Chengmou. 2008. Chemiluminescence determination of organophosphorus pesticide chlorpyrifos in vegetable. Analytical Letters 41(8): 1375–1386. Li, Li; Zhiqiang, Zhou; Canping, Pan; Chuanfan, Qian; Shuren, Jiang; Fengmao, Liu. 2007. Determination of organophosphorus pesticides in soybean oil, peanut oil and sesame oil by low-temperature extraction and GCFPD. Chromatographia 66(7–8):625–629. Liang, Ying; Liu, Xian Jin; Liu, Yuan; Yu, Xiang Yang; Fan Ming, Tao. 2008. Synthesis of three haptens for the class-specific immunoassay of O,O-dimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity. Analytica Chimica Acta 615(2):174–183. Liu, Yongmei; Wang, Zhihua; Chu, Xiaogang. 2005. Determination of 50 organophosphorus pesticides in unpolished rice sample by gas chromatographynitrogenphosphorus detection with gel permeation chromatography cleanup. Fenxi Huaxue 33(6): 808–810. Lord, K.A.; Potter, C. 1950. Mechanism of action of organophosphorus compounds as insecticides. Nature 166:893–894. Mahjoubi-Samet, A.; Hamadi, F.; Soussia, L.; Fadhel, G.; Zeghal, N. 2005. Dimethoate effects on thyroid function in suckling rats. Annales d′endocrinologie, 66(2):96–104. Marco, María Pilar; Gee, Shirley; Hammock, Bruce D. 1995. Immunochemical techniques for environmental analysis. I. Immunosensor. Trends in Analytical Chemistry 14(7):341–350. McKinlay, R.; Plant, J.A.; Bell, J.N.B.; Voulvoulis, N. 2008. Endocrine disrupting pesticides: Implications for risk assessment. Environment International 34(2):168–183. Mezcua, Milagros; Ferrer, Carmen; García-Reyes, Juan F.; Martínez-Bueno, María Jesus; Albarracín, Micaela; Claret, María; Fernández-Alba, Amadeo R. 2008. Determination of selected non-authorized insecticides in peppers by liquid chromatography time-of-flight mass spectrometry and tandem mass spectrometry. Rapid Communications in Mass Spectrometry 22(9): 1384–1392. Mezcua, Milagros; Ferrer, Carmen; García-Reyes, Juan F.; Martínez-Bueno, María Jesús; Albarracín, Micaela; Sigrist, Mirna; Fernández-Alba, Amadeo R. 2009a.

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Determination of selected non-authorized insecticides in peppers by gas chromatography/mass spectrometry and tandem mass spectrometry. Food Chemistry 112(1):221–225. Mezcua, Milagros; Malato, Octavio; García-Reyes, Juan F.; Molina-Díaz, Antonio; Fernández-Alba, Amadeo R. 2009b. Accurate mass databases for large screening of pesticides in food by liquid chromatography timeof-flight mass spectrometry. Analytical Chemistry 81(3):913–929. Min, Liu; Yuki, Hashi; Yuanyuan, Song; Jin-Ming, Lin. 2005. Simultaneous determination of carbamate and organophosphorus pesticides in fruits and vegetables by liquid chromatography-mass spectrometry. Journal of Chromatography A 1097(1):183–187. Moris, Philip; Alexandre, Isabelle; Roger, Marc; Remacle, José. 1995. Chemiluminescence assays of organophophorus and carbamate pesticides. Analytica Chimica Acta 302(1):53–59. Mulchandani, Ashok; Chen, Wilfred; Mulchadani, Priti; Wang, Joseph; Rogers, Kim R. 2001. Biosensors for direct determination of organophosphate pesticides. Biosensors and Bioelectronics 16(4–5):225–230. Nunes, Gilvanda Siva; Toscano, Ilda Antonieta; Barceló, Damia. 1998. Analysis of pesticides in food and environmental samples by enzyme-linked immunosorbent assays. Trends in Analytical Chemistry 17(2):79–87. Okubo, T.; Yokoyama, Y.; Kano, K.; Soya, Y.; Kano, I. 2004. Estimation of estrogenic and antiestrogenic activities of selected pesticides by MCF-7 cell proliferation assay. Archives of Environmental Contamination and Toxicology 46(4)445–453. Ouyang, Zheng; Noll, Robert J.; Cooks, R. Graham. 2009. Handheld miniature ion trap mass spectrometers. Analytical Chemistry 81(7):2421–2425. Pang, Guo-Fang; Liu, Yong-Ming; Fan, Chun Lin; Zhang, Jin-Jie; Cao, Yan-Zhong; Li, Xue-Min; Li, Zeng-Yin; Wu, Yan-Pin; Guo, Tong-Tong. 2006. Simultaneous determination of 405 pesticide residues in grain by accelerated solvent extraction then gas chromatography-mass spectrometry or liquid chromatography-tandem mass spectrometry Analytical and Bioanalytical Chemistry 384(5):1366–1408. Periasamy, Arun Parkash; Umasankar, Yogeswaran; Chen, Shen Ming. 2009. Nanomaterialsacetylcholinesterase enzyme matrices for organophosphorus pesticides electrochemical sensors: A review. Sensors 9(6):4035–4055. Picó, Yolanda; Blanco, Cristina; Font, Guillermina. 2004. Environmental and food applications of LCtandem mass spectrometry in pesticide-residue analysis: An overview. Mass Spectrometry Reviews 23(1):45–85. Psillakis, E.; Kalogerakis, N. 2002. Developments in single-drop microextraction. Trends in Analytical Chemistry 21(1):54–64. Richard, S.; Moslemi, S.; Sipahutar, H.; Benachour, N.; Seralini, G.-E. 2005. Differential effects of glyphosate and roundup on human placental cells and aromatase. Environmental Health Perspectives 113(6):716–720. Ridgway, Kathy; Lalljie, Sam P.D.; Smith, Roger M. 2007. Sample preparation techniques for the determi-

218

Analysis of Endocrine Disrupting Compounds in Food

nation of trace residues and contaminants in food. Journal of Chromatography A 1153(1–2):36–53. Rissato, Sandra R.; Galhiane, Mario S.; de Almeida, Marcos V.; Gerenutti, Marli; Apon, Bernhard M. 2007. Multiresidue determination of pesticides in honey samples by gas chromatography-mass spectrometry and application in environmental contamination. Food Chemistry 101(4):1719–1726. Roda, Aldo; Rauch, Pavel; Ferri, Elida; Girotti, Stefano; Ghini, Severino; Carrea, Giacomo; Bovara, Roberto. 1994. Chemiluminescent flow sensor for the determination of paraoxon and aldicarb pesticides. Analytica Chimica Acta 294(1):35–42. Sancho, Juan V.; Pozo, Óscar J.; Hernández, Félix. 2004. Liquid chromatography and tandem mass spectrometry: A powerful approach for the sensitive and rapid multiclass determination of pesticides and transformation products in water. The Analyst 129(1): 38–44. Schöning, Michael J.; Arzdorf, Michael; Mulchandani, Priti; Chen, Wilfried; Mulchandani, Ashok. 2003. Towards a capacitive enzyme sensor for direct determination of organophosphorus pesticides: Fundamental studies and aspects of development. Sensors 3(6):119–127. Shu, You-Ren.; Su, An-Kai; Liu, Ju-Tsung; Lin, ChengHuang. 2006. Screening of nerve agent degradation products by MALDI-TOFMS. Analytical Chemistry 78(13):4697–4701. Singh, A.K.K. 2002. Acute effects of acephate and methadimophos and interleukin-1 on corticotrophinreleasing factor (CRF) synthesis in and release from the hypothalamus in vitro. Comparative biochemistry and physiology-Part C-toxicology and pharmacology. 132(1):9. Song, Zhenghua; Hou, Shuang; Zhang, Ni. 2002. A new green analytical procedure for monitoring subnanogram amounts of chlorpyrifos on fruits using flow injection chemiluminescence with immobilized reagents. Journal of Agricultural and Food Chemistry 50(16):4468–4474. Steiner, Wes E.; Klopsch, Steve J.; English, William A.; Clowers, Brian H.; Hill, Herbert H. 2005. Detection of chemical warfare agent simulant in various aerosol matrixes by ion mobility time-of-flight mass spectrometry. Analytical Chemistry 77(15):4792–4799. Trojanowicz, Marek; Hitchman, Michael. 1996. Determination of pesticides using electrochemical biosensors. Trends in Analytical Chemistry 15(1): 38–45.

Tsoutsi, C.; Konstantinou, I.; Hela, D.; Albanis, T.A. 2006. Screening method for organophosphorus insecticides and their metabolites in olive oil samples based on headspace solid-phase microextraction coupled with gas chromatography. Analytica Chimica Acta 573–574(1):216–222. Venter, Andre; Nefliu, Marcela; Cooks, R. Graham. 2008. Ambient desorption ionization mass spectrometry. Trends in Analytical Chemistry 27(4):284–290. Vercauteren, Jordy; Peres, Christophe; Devos, Christophe; Sandra, Pat; Vanhaecke, Frank; Moens, Luc. 2001. Stir bar sorptive extraction for the determination of ppq-level traces of organotin compounds in environmental samples with thermal desorption capillary gas chromatography-ICP mass spectrometry. Analytical Chemistry 73(7):1509–1514. Vinggaard, A.; Hass, U.; Dalgaard, M.; Andersen, H.R.; Bonefeld-Jorgensen, E.; Christiansen, S.; Laier, P.; Poulsen, M.E. 2005. Prochloraz: An imidazole fungicide with multiple mechanisms of action. International Journal of Andrology 29(1):186–192. Wang, Jianning; Zhang, Chao; Wang, Haixia; Yang, Fengzhen; Zhang, Xinrong. 2001. Development of a luminol-based chemiluminescence flow-injection method for the determination of dichlorvos pesticide. Talanta 54(6):1185–1193. Weinrich, Luise; Popp, Peter; Köller, Gábor; Breuste, Jürgen. 2001. Determination of organochlorine pesticides and chlorobenzenes in strawberries by using accelerated solvent extraction combined with sorptive enrichment and gas chromatography/mass spectrometry. Journal of AOAC International 84(6): 1194–1201. Wenmin, Liu; Yuan, Hu; Jinghong, Zhao; Yuan, Xu; Yafeng, Guan. 2005. Determination of organophosphorus pesticides in cucumber and potato by stir bar sorptive extraction. Journal of Chromatography A 1095(1):1–7. Wuilloud, Rodolfo G.; Shah, Monika; Kannamkumarath, Sasi S.; Altamirano, Jorgelina C. 2005. The potential of inductively coupled plasma-mass spectrometric detection for capillary electrophoretic analysis of pesticides. Electrophoresis 26(7–8): 1598–1605. Zambonin, Carlo G.; Quinto, Maurizio; De Vietro, Nicoletta; Palmisano, Francesco. 2004. Solid-phase microextraction-gas chromatography mass spectrometry: A fast and simple screening method for the assessment of organophosphorus pesticide residues in wine and fruit juices. Food Chemistry 86(1–2): 269–274.

Chapter 7 Phytoestrogens Ashok K. Singh and Leo M.L. Nollet

Introduction Phytoestrogens or naturally occurring endocrine-disrupting chemicals (EDCs) are a diverse group of naturally occurring nonsteroidal plant compounds. They are included in foods such as soybeans, apples, cherries, wheat, and peas (Delclos et al. 2009; Dixon 2004; Sirtori et al. 2005; Moon et al. 2006). Coumestans, prenylated flavonoids, isoflavones, and lignans are the best known phytoestrogens.

Phytoestrogens Isoflavones Isoflavones (Figure 7.1) comprise a large and very distinctive subclass of phytoestrogens encompassing several structurally and synthetically related classes, such as flavones, flavonols (3-hydroxyflavones), anthocyanins, flavanones, isoflavonoids (isoflavones, coumestans), and chalcones. Isoflavonoids have the phenyl ring (B-ring) attached at the 3 position rather than at the 2 position of the heterocyclic ring and exhibit greater structural variation and greater frequency of isoprenoid substitution. Isoflavones are isomeric with the more widely occurring flavones. For example, genistein is derived biosynthetically by an aryl migration from the same chalcone precursor as that which gives rise to Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

the flavone apigenin. Isoflavones such as genistein, daidzein, biochanin A, and formononetin have been most thoroughly investigated with regard to estrogenicity (Whitten et al. 1997). Genistein (4′,5,7-trihydroxyisoflavone) is the most active principle with the highest binding affinity for the estrogen receptor (Shutt and Cox 1972). Its methoxy derivative, biochanin A, does not bind to the estrogenic receptor in vitro but is estrogenic in vivo (Braden et al. 1967; Miksicek 1994). Daidzein (4′,7-dihydroxyisoflavone) has a higher binding affinity for the estrogen receptor than its methoxy derivative formononetin (Shutt and Cox 1972). This suggests that methylation could be the mechanism through which the estrogenic potency of isoflavones is reduced (Bickoff et al. 1962). The 5-hydroxy group of genistein may account for the differential potency between genistein and daidzein (Bickoff et al. 1962). Concentration of isoflavones in different food types is shown in Figure 7.2. Apart from their estrogenic effects, isoflavones also exhibit antiestrogenic effects in vivo. Estrogenic property of synthetic or natural EDCs is counteracted by administered isoflavonoids (Folman and Pope 1966, 1969; Kitts et al. 1983). At relatively higher concentrations, isoflavonoids may compete effectively with endogenous mammalian estrogens and prevent estrogen-stimulated growth in mammals (Adlercreutz 1995). Such high phytoestrogen levels are achieved by regular phytoestrogen consumption. Earlier studies (Kuiper et al. 1996; Mosselman et al. 1996; 219

O 3

O

8

2

7 6

4

5

O

R1

1′

H

4′

O

R1 H H OH OH

R2 H CH3 H CH3

Counmestrol H 4′-Methoxycoumestrol H Repensol OH Trifoliol OH

H CH3 H CH3

H Daidzein Formononetin Genistein Biochanin A

O R2 4′

O H

6 5

4

7 8

1 2

O

O

Estradiiol H

1′ 2′ 3

R1 O H CH3 O

H H

H

O

Figure 7.1. Structure of representative isoflavones.

A

B

Bread, rye Black licorice Black bean souce Beer Coffee, decaf Tea, black Wine, white Tea, green Wine, red Lentils Hazel nuts Cashews Walnuts Chestnuts Pistachios Watermelon Raspberry Strawberry Peaches Dried prunes Cabbage Broccoli Collards Green beans Winter squash Garlic Soy bean sprouts

Lignan Phytoestrogen

3

1

×

×

6

6

10

10

00 12 00 10 0 80

0 60

0 40

0 20

Milk, cow Watermelon Coffee, regular Corn Blueberry Onion Peanuts Green beans Almonds Olive oil Chestnuts Sunflower seeds Dried dates Alfalfa sprouts Dried apricots Mung bean sprouts Garlic Hummus Soy milk Multigrain bread Flax bread Sesame seeds Soy yogurt Tofu Soy beans Flax seed

Concentraion μg/100g

4

3

2

1

×

×

×

×

6

10

6

10

6

10

6

10

00 10 0 50 0 00

Figure 7.2. (A) Quantification of isoflavones in different food samples. Flaxseed, soybean, and tofu contain high levels of isoflavones. Food processing decreases isoflavone contents. (B) Comparison of phytoestrogen and lignan contents in different food items. Soybeans and black beans contain mostly isoflavones, whereas other food items studied contained comparable levels of total phytoestrogens and total lignans.

220

Phytoestrogens

221

Table 7.1. Effects of endocrine disruptors on different indices of endocrine toxicity. Toxins Dieldrin Endosulfan Methiocarb Primicarb Propamocarb Fenarimol Prochloraz Genistein Equol Bisphenol A 3-hydroxy-benzophenone

Prop.

Cell Pro.

ER-Tra.

AR-Tra.

Aromatase

OC OC CAR CAR CAR FUN FUN PHYTO PHYTO SYN IND

I I I NC NC I D I NC I N/A

I I I I I I D I NC I I

D D D NC NC I D D D D D

NC D NC I I D D I NC I N/A

Prop., toxin’s properties; Cell Pro., effects of toxins on cell proliferation in vitro; ER-Tra., effects of toxins on estrogen receptor (ER) transactivation; AR-Tra., effects of toxins on androgen receptor (AR) transactivation; Aromatase, effects of toxins on aromatase activity; OC, organochlorine insecticides; CAR, carbamates; FUN, fungicides; PHYTO, phytoestrogens; SYN, synthetic, IND, industrial; I, increase; D, decrease; NC, no change; N/A, data not available.

Byers et al. 1997) have identified two types of estrogen receptors (ERs), ERα, which induces estrogenic effects, and ERβ, which induces antiestrogenic activity. Both coumestrol and genistein exhibit a significantly higher affinity for ERβ protein than for ERα (Cassidy 1996), which may account for antiestrogenic effects of the phytoestrogens. There are two different forms of the estrogen receptor, ERα and ERβ, each encoded by a separate gene, ESR1 and ESR2, respectively. Hormone-activated estrogen receptors form dimers, and because the two forms are coexpressed in many cell types, the receptors may form ERα (αα) or ERβ (ββ) homodimers or ERαβ (αβ) heterodimers (Huang et al. 2004). ERα and ERβ show significant overall sequence homology, and both are composed of seven domains; however, ERβ may lack the AF1 site. Phytoestrogens have affinity to ERα and ERβ. A number of phytoestrogens exhibited greater affinity for ERβ than for ERα. It is postulated that ERα and ERβ have different or even opposite biologic actions (Lindberg et al. 2003). ERβ may negatively regulate cellular proliferation and have a protective role in normal breast (Hilakivi-Clarke et al. 2002). ERα and ERβ can heterodimerize, and when coexpressed in the same cell, ERβ exerts an inhibitory action on ERα-mediated

RO

3

2

1

7

8

9

OH

4

HO

6

8′

7′

5

OR2 9′

1′ 2′

6′

3′

5′

OR1

4′

OH R 1. 3′-DemethylisolariciresinolCH3 9′-hydroxyisopropylether

R1

R2

H

C

OH

2. 3-Demethylisolariciresinol

H

CH3 CH3 H

3. Isolariciresinol

CH3

CH3

CH3

H

Figure 7.3. Structures of different lignans. From Erdemoglu et al. (2003), with permission.

gene expression (Matthews and Gustafsson 2003). Table 7.1 gives an overview of the effects of genistein, among other endocrine disruptors, on different indices of endocrine toxicity.

Dietary lignans Dietary lignans are found mainly in woody tissues (Figure 7.3) (Ayres et al. 1996; Ayres and Loike 1990) and, to some extent, in leaves and flowers (Ayres and Loike 1990). In general, lignans are a union of two cinnamic acid residues (2, 3-dibenzylbutane structure)

222

Analysis of Endocrine Disrupting Compounds in Food

1 (CH2)4 O H3C

OH

OH COOH

H3C

(CH2)2

O

2

(CH2)4

OH COOH (CH2)4

HO OH 1: 10,13-Oxy- 9, 12-Dihydroxyoctadeconoic acid 2: 9,12-Oxy-10, 13-Dihydroxyoctadeconoic acid OH HO O

OH 9, 10-Dihydroxy-12-octadeconoic acid OH

HO O

OH 12, 13-Dihydroxy- 9-octadeconoic acid

Figure 7.4. Structure of corn-based endocrine disruptors.

or their biogenetic equivalents. The majority of the 200 or more naturally occurring lignans (Ayres and Loike 1990) occur as aglycosides (unconjugated form) and as glycosides (Ayres and Loike 1990; Ward 1993, 1995). Apart from plants, lignans have also been identified in humans and animals. Serum, urine, bile, and seminal fluids of humans and animals contain enterolactone (ENL) and enterodiol (END) (Setchell and Adlercreutz 1988; Stitch et al. 1980; Setchell et al. 1980b). The mammalian-derived lignans possess phenolic hydroxyl groups only in the meta position of the aromatic rings. The dietary precursors of these lignans are secoisolariciresinol (Setchell et al. 1980a) and matairesinol (MAT) (Borriello et al. 1985). Although lignans structurally resemble estrogens, experimental evidence concerning hormonal actions of lignans is conflicting.

Corn products as endocrine disruptors Ground corncob animal bedding and corn food products contain substances that disrupt endocrine function (Markaverich et al. 2005, 2007). The disruptors were

identified as isomeric mixtures of tetrahydrofurandiols (THF-diols; 9,12-oxy-10, 13-dihydroxyoctadecanoic acid and 10,13oxy-9,12-dihydroxyoctadecanoic acid) and leukotoxindiols (LTX-diols; 9,10-dihydroxy12-octadecenoic acid and 12,13-dihydroxy9-octadecenoic acid) (Figure 7.4). The authentic compounds blocked sexual behavior in male rats and estrous cyclicity in female rats at oral doses of 2 ppm. Thus corn, cornderived products, and sweeteners may contain potential estrogenic and other endocrinedisrupting substances that may present health risks in humans. The widespread use of corn oil, high-fructose corn syrup, and other sweeteners in human diets lends a sense of urgency to further investigation.

Metabolism Isoflavonoids and lignans show similar patterns of metabolism in animals (Price and Fenwick 1985) and human subjects (Ohtomo et al. 2008; Adlercreutz 2007; Adlercreutz et al. 1987, 1991; D’Alessandro et al. 2005; Lampe et al. 1998). In humans, isoflavone and lignan glycosides are hydrolyzed by

Phytoestrogens

gastric acid (Xu et al. 1995) and undergo enzymatic hydrolysis by intestinal microflora (Setchell and Adlercreutz 1988). Intestinal bacterial glycosidases cleave the sugar moieties and release the biologically active aglycones, which can be further biotransformed by bacteria to specific metabolites (Day et al. 2000; Walle 2004). Biochanin A is converted to genistein, which is further metabolized to dihydrogenistein, 6′-OH- desmethylangolensin, and p-ethylphenol. Daidzin is metabolized to equol and 3- or 6-OH equol (Soucy et al. 2006; D’Alessandro et al. 2005; Peterson et al. 1998). A study of the bioavailability and metabolism of daidzein, genistein, and glycitein (Zhang et al. 1999) showed that the average 48-h urinary excretion of glycitein, daidzein, and genistein was approximately 55%, 46%, and 29% of the dose ingested, respectively.

Analysis of phytoestrogens Solid-phase extraction (SPE) and high performance liquid chromatography mass spectrometry (HPLC-MS) or gas chromatography mass spectrometry (GC-MS) techniques have been used in determining estrogens and isoflavones extracted from biological and plant samples (Singh 2007; Benlhabib et al. 2004; Holder et al. 1999). Satterfield et al. (2001) have developed a rapid preconcentration of the analytes that significantly improved detection sensitivity (the lowest reported detectable levels of daidzein and genistein were 25.4 and 2.70 pg/mL, respectively). Barnes et al. (1998a, b) developed a microbore HPLC electrospray ionization mass spectrometry (HPLC-ESI-MS) positive ionization method that used API and yielded a detection limit of 0.2 mg/kg for daidzein and 0.7 mg/kg for genistein. Recently, Benlhabib et al. (2004) developed a LC-MS method for analysis of isoflavones extracted from plant extracts and tissue samples. The chromatographic design separated and quantified conjugated and simple analytes. Similarly, Cimino et al.

223

(1999) reported a LC-MS method to estimate urinary concentration of genistein and daidzein and their sulfate and glucuronide conjugates in urine samples. In rat urine, 52 ± 4% and 26 ± 4% of genistein existed as aglycone and sulfate conjugate, respectively. In human urine, 0.36% and 9% of genistein in rat urine existed as aglycone and sulfate conjugate, respectively. Some investigators (ValentinBlasini et al. 2000; Coward et al. 1996) have used enzymatic hydrolysis followed by solvent extraction to recover the phytoestrogen aglycones (phenolphthalein βglucuronide, 4-methylumbelliferone sulfate, and apigenin were added to each sample as internal standards). This technique simplifies the analysis of phytoestrogens because it removes the necessity for gradient chromatography (Horn-Ross et al. 1997, 2000). For structural information, triple quadrupole LC-MS has been used (Barnes et al. 1998a, b). Studies have shown that an ion at m/z 133 is diagnostic for genistein and daidzein but not for their flavonoid isomers, such as apigenin (Barnes et al. 1998a, b). Table 7.2 gives a comparison of extraction techniques and methods. Figure 7.5 shows GC spectrograms of saponified lignan.

Application of mass spectrometry in analysis of phytoestrogens Mass spectral techniques have taken the analysis and identification of the bioflavonoids and toxicants giant steps forward. Mass spectrometers coupled to a GC or HPLC create versatile equipment able to simultaneously analyze structurally diverse chemicals. Because of the development of a mass spectral search for identifying chemicals, it is possible to study the metabolism and fate of toxicants. It is probable that many new bioactive metabolites will be discovered at the tissue target sites. An example of a separation using HPLC-MS/MS is given in Figure 7.6.

224 Isoflavones Gi, Ge, and De Di, Gi, Gly, De, Ge, Gle, MDi, MGi, MGly, ADi, AGi, and AGly De and Ge Di, Gi, De, and Ge Di, Gi, Gly, and MGi Di, Gi, Gly, De, Ge, Gle, MDi, MGi, MGly, ADi, AGi, and AGly Di, Gi, De, and Ge Di, Gi, Gly, Ononin, De, Gle, and Ge Gi, MGi, AGi, and Ge De and Ge

Compared Techniques & Methods

Relative Yield (%)

98–100/88–100 100/93/16/71/69

49/14/64/100 22/68/100

PLE/UAE/Soxhlet/PLE + UAE UAE/Soxhlet/PLE + UAE PLE/Stirring UAE/UHOM/SFE/PLE/Soxhlet

26/100 87/100 100/85–100 100/93/68/71/66/70

28/100/68 74/100

SFE/Stirring SFE/Stirring UAE/Stirring PLE/UAE/Soxhlet/Shaker/Vortex/Stirring

SFE/UAE/Soxhlet SFE/Shaking

References

Bajer et al. 2007 Rostango et al. 2007

Klejdus et al. 2004 Klejdus et al. 2004

Franke et al. 1994 Zuo et al. 2008 Rostagno et al. 2003 Luthria et al. 2007

Rostagno et al. 2002 Araújo et al. 2007

De, diadzein; Ge, genistein; Gle, glycitein; Di, daidzin; Gi, genistin; Gly, glycitin; MDi, manolyl daidzin; MGi, malonyl genistin; MGly, malonyl glycitin; ADi, acetyl daidzin; AGi, acetyl genistin; AGly, acetyl glycitin; UHOM, ultrasonic homogenizer; SFE, supercritical fluid extraction; UAE, ultrasound-assisted extraction; SWE, superheated water extraction; PLE, pressurized liquid extraction; SPE: solid-phase extraction. From Rostagno et al. (2009). Modified with permission.

Soy flour Soy flour

Soy bits Soy bits

Soybean hypocotyls Soybean meal Soybeans Soybeans

Soy flour Soybean cake

Sample

Table 7.2. Relative comparison of extraction techniques and methods.

(A) 100

401.1

Relative Abundance

415.2 80 60 417.2 40 399.2 20 0

433.0

345.2 385.2 320

360

(B) 100

400

531.1 553.0 515.3 537.0 483.1 501.0

440 480 m/z

520

560

600

417.2

Relative Abundance

80 415.2

60

553.0 40 399.2

20

531.0 537.0 515.1521.2

316.2 347.2371.2 0

320

360

400

440 480 m/z

520

560

600

Figure 7.5. Full-scan ESI-MS spectra of the methanol extracts lignans from (A) Schisandra chinensis fruits and (B) Schisandra sphenanthera fruits. From Xing Huang et al. (2008), with permission.

255 199, 153 Daidzein 271 153, 91

Genistein

242 143, 97

Bisphenol-d16

227 133, 112 Bisphenol 295 145, 143 Ethynylestradiol 159, 183 205 133, 117 4-t-Octylphenol 219

133

205 106 12 13 14 15 16 17 18

Nonylphenol 4-n-Octylphenol 12 13 14 15 16 17 18

Figure 7.6. Chromatographic separation of isoflavones and polyphenols extracted from spiked water and plasma samples extracted using a C18 SPE. Samples were analyzed using HPLC-MS/MS (Singh 2007).

225

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References Adlercreutz H. (1995) Phytoestrogens: Epidemiology and a possible role in cancer protection. Environ Health Perspect. 103, 103–112. Adlercreutz H. (2007) Lignans and human health. Crit Rev Clin Lab Sci. 44, 483–525. Adlercreutz H. Honjo H. Higashi A. Fotsis T. Hamalainen E. Hasegawa T. Okada H. (1991) Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am J Clin Nutr. 54, 1093–1100. Adlercreutz H. Hockerstedt K. Bannwart C. Bloigu S. Hamalainen E. Fotsis T. Ollus A. (1987) Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG). J Steroid Biochem. 27, 1135–1144. Araújo JMA. Silva MV. Chaves JBP. (2007) Supercritical fluid extraction of daidzein and genistein isoflavones from soybean hypocotyl after hydrolysis with endogenous B-glucosidases. Food Chem. 105, 266–272. Ayres S. Tang M. Subbiah MT. (1996) Estradiol-17β as an antioxidant: Some distinct features when compared with common fat-soluble antioxidants. J Lab Clin Med. 128, 367–375. Ayres DC. Loike JD. (1990) Lignans: Chemical, Biological and Clinical Properties (Chemistry and Pharmacology of Natural Products). Cambridge, UK: Cambridge University Press. Bajer T. Adam M. Galla L. Ventura K. (2007) Comparison of various extraction techniques for isolation and determination of isoflavonoids in plants. J Sep Sci. 30, 122–127. Barnes KA. Smith RA. Williams K. Damant AP. Shepherd MJ. (1998a) A microbore high performance liquid chromatography/electrospray ionization mass spectrometry method for the determination of the phytoestrogens genistein and daidzein in comminuted baby foods and soya flour. Rapid Commun Mass Spectr. 12, 130–138. Barnes S. Wang CC. Kirk M. Smith-Johnson M. Coward L. Barnes NC. Vance G. Boersma B. (1998b) HPLCmass spectrometry of isoflavonoids in soy and the American groundnut, Apios americana. Proc Soc Exp Biol Med. 217, 254–262. Benlhabib E. Baker JI. Keyler DE. Singh AK. (2004) Quantitative analysis of phytoestrogens in kudzu-root, soy, and spiked serum samples by high-pressure liquid chromatography. Biomed Chromatogr. 18, 367–380. Bickoff EM. Livingston AL. Hendrickson AP. Booth AN. (1962) Relative potencies of several estrogen-like compounds found in forages. J Agric Food Chem. 10, 410–415. Borriello SP. Setchell KDR. Axelson M. Lawson AM. (1985) Production and metabolism of lignans by the human fecal flora. J Appl Bacteriol. 58, 37–43. Braden AWH. Hart NK. Lamberton JA. (1967) The estrogenic activity and metabolism of certain isoflavones in sheep. Aust J Agric Res. 18, 335–348. Byers M. Kuiper GGJM. Gustafsson JÅÅ. Park-Sarge O-K. (1997) Estrogen receptor-β mRNA expression in

rat ovary: Down-regulation by gonadotropins. Mol Endocr. 11, 172–182. Cassidy A. (1996) Physiological effects of phytooestrogens in relation to cancer and other human health risks. Proc Nutr Soc. 55, 399–417. Cimino CO. Shelnutt SR. Ronis MJ. Badger TM. (1999) An LC-MS method to determine concentrations of isoflavones and their sulfate and glucuronide conjugates in urine. Clin Chim Acta. 287, 69–82. Coward L. Kirk M. Albin N. Barnes S. (1996) Analysis of plasma isoflavones by reversed-phase HPLC-multiple reaction ion monitoring-mass spectrometry. Clin Chim Acta. 247, 121–142. D’Alessandro TL. Boersma-Maland BJ. Peterson TG. Sfakianos J. Prasain JK. Patel RP. Darley-Usmar VM. Botting NP. Barnes S. (2005) Metabolism of phytoestrogen conjugates. Methods Enzymol. 400, 316–342. Day AJ. Canada FJ. Diaz JC. Kroon PA. Mclauchlan R. Faulds CB. Plumb GW. Morgan MRA. Williamson G. (2000) Dietary flavonoid and isoflavonoid glycosides are hydrolyzed by the lactase site of lactase phlorizin hydrolase. FEBS Letters. 468, 166–170. Delclos KB. Weis CC. Bucci TJ. Olson G. Mellick P. Sadovova N. Latendresse JR. Thorn B. Newbold RR. (2009) Overlapping but distinct effects of genistein and ethinyl estradiol (EE(2)) in female SpragueDawley rats in multigenerational reproductive and chronic toxicity studies. Repr Tox. 27, 117–132. Dixon RA. (2004) Phytoestrogens. Ann Rev Plant Biol. 55, 225–261. Erdemoglu N. Sener B. Ozcan Y. Ide S. (2003) Structural and spectroscopic characteristics of two new dibenzylbutane type lignans from Taxus baccata L. J Mole Struct. 655, 459–466. Folman Y. Pope GS. (1966) The interaction in the immature mouse of potent oestrogens with coumestrol, genistein, and other utero-vaginotrophic compounds of low potency. J Endocrinol. 34, 215–225. Folman Y. Pope GS. (1969) Effect of norethisterone acetate, dimethylstilboestrol, genistein, and coumestrol on uptake of [3H]oestradiol by uterus, vagina, and skeletal muscle of immature mice. J Endocrinol. 44, 213–218. Franke A. Custer LJ. Cerna CM. Narala KK. (1994) Quantitation of phytoestrogens in legumes by HPLC. J Agric Food Chem. 42, 1905–1913. Hilakivi-Clarke L. Cho E. Cabanes A. DeAssis S. Olivo S. Helferich W. Lippman ME. Clarke R. (2002) Dietary modulation of pregnancy estrogen levels and breast cancer risk among female rat offspring. Clin Cancer Res. 8, 3601–3610. Holder CL. Churchwell MI. Doerge DR. (1999) Quantification of soy isoflavones, genistein and daidzein, and conjugates in rat blood using LC/ES-MS. J Agr Food Chem. 47, 3764–3770. Horn-Ross PL. Barnes S. Kirk M. Coward L. Parsonnet J. Hiatt RA. (1997) Urinary phytoestrogen levels in young women from a multiethnic population. Cancer Epidemiol Biomarkers Prev. 6, 339–345. Horn-Ross PL. Lee M. John EM. Koo J. (2000) Sources of phytoestrogen exposure among non-Asian women in California, USA. Cancer Causes Control. 11, 299– 302.

Phytoestrogens

Huang J. Li X. Yi P. Hilf R. Bambara RA. Muyan M. (2004) Targeting estrogen responsive elements (EREs): Design of potent transactivators for EREcontaining genes. Mol Cell Endocrinol. 218, 65–78. Huang X. Song F. Liu Z. Liu S. (2008) Structural characterization and identification of dibenzocyclooctadiene lignans in Fructus schisandrae using electrospray ionization ion trap multiple-stage tandem mass spectrometry and electrospray ionization and Fourier transform ion cyclotron resonance multiple-stage tandem mass spectrometry. Anal Chim Acta. 615, 124–135. Kitts WD. Newsome FE. Runeckles VC. (1983). The estrogenic and antiestrogenic effects of coumestrol and zearalanol on the immature rat uterus. Can J Anim Sci. 63, 823–834. Klejdus B. Mikelová R. Adam V. Zehnálek J. Vacek J. Kizek R. Kubán V. (2004) Liquid chromatographic– mass spectrometric determination of genistin and daidzin in soybean food samples after accelerated solvent extraction with modified content of extraction cell. Anal Chim Acta. 517, 1–11. Kuiper GG. Enmark E. Pelto-Huikko M. Nilsson S. Gustafsson JA. (1996) Cloning of a novel receptor expressed in rat prostate and ovary. PNAS USA. 93, 5925–5930. Lampe J. Karr SC. Hutchins AM. Slavin JL. (1998) Urinary equol excretion with a soy challenge: Influence of habitual diet. Proc Soc Exp Biol Med. 217, 335– 339. Lindberg MK. Movérare S. Skrtic S. Gao H. DahlmanWright K. Gustafsson JA. Ohlsson K. (2003) Estrogen receptor (ER)-β reduces ERα-regulated gene transcription, supporting a “ying yang” relationship between ERα and ERβ in mice. Mol Endocr. 17, 203–208. Luthria DL. Biswas R. Natarajan S. (2007) Comparison of extraction solvents and techniques used for the assay of isoflavones from soybean. Food Chem. 105, 325–333. Markaverich BM. Alejandro M. Thompson T. Mani S. Reyna A. Portillo W. Sharp J. Turk J. Crowley JR. (2007) Tetrahydrofurandiols (THF-diols), leukotoxindiols (LTX-diols), and endocrine disruption in rats. Environ Health Persp. 115, 702–708. Markaverich BM. Crowley JR. Alejandro MA. Shoulars K. Casajuna N. Mani S. Reyna A. Sharp J. (2005) Leukotoxin diols from ground corncob bedding disrupt estrous cyclicity in rats and stimulate MCF-7 breast cancer cell proliferation. Environ Health Persp. 113, 1698–1704. Matthews J. Gustafsson JÅ. (2003) Estrogen signalling: A subtle balance between ERα and ER4β. Mol Interventions. 3, 281–292. Miksicek RJ. (1994) Interaction of naturally occurring nonsteroidal estrogens with expressed recombinant human estrogen receptor. J Steroid Biochem Mol Biol 49, 153–160. Moon YJ. Wang X. Morris ME. (2006) Dietary flavonoids: Effects on xenobiotic and carcinogen metabolism. Toxicol In Vitro. 20, 187–210. Mosselman S. Polman J. Dijkema R. (1996) ER beta: Identification and characterization of a novel human estrogen receptor. FEBS Letters. 392, 49–53.

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Ohtomo T. Uehara M. Penalvo JL. Adlercreutz H. Katsumata S. Suzuki K. Takeda K. Masuyama R. Ishimi Y. (2008) Comparative activities of daidzein metabolites, equol and O-desmethylangolensin, on bone mineral density and lipid metabolism in ovariectomized mice and in osteoclast cell cultures. Eur J Nutr. 47, 273–279. Peterson TG. Ji GP. Kirk M. Coward L. Falany CN. Barnes S. (1998) Metabolism of the isoflavones genistein and biochanin A in human breast cancer cell lines. Am J Clin Nutr. 68, 1505S–1511S. Price KR. Fenwick GR. (1985). Naturally occurring estrogens in foods: A review. Food Addit Contam. 2, 73–106. Rostagno MA. Villares A. Guillamon E. Garcia-Lafuente A. Martinez JA. (2009) Sample preparation for the analysis of isoflavones from soybeans and soy foods. J Chromatogr A. 1216, 2–29. Rostagno MA. Palma M. Barroso CG. (2003) Ultrasoundassisted extraction of soy isoflavones. J Chromatogr A. 1012, 119–128. Rostagno MA. Araújo JMA. Sandi D. (2002) Supercritical fluid extraction of isoflavones from soybean flour. Food Chem. 78, 111–117. Satterfield M. Black DM. Brodbelt JS. (2001) Detection of the isoflavone aglycones genistein and daidzein in urine using solid-phase microextraction-high-performance liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr B Biomed Sci Appl. 759, 33–41. Setchell KDR. Lawson AM. Mitchell FL. Adlercreutz H. Kirk DN. Axelson M. (1980a) Lignans in man and animal species. Nature. 287:740–742. Setchell KDR. Bull R. Adlercreutz H. (1980b) Steroid excretion during the reproductive cycle and in pregnancy of the vervet monkey (Cercopithecus aethiopus pygerythrus). J Steroid Biochem. 12, 375–384. Setchell KDR. Adlercreutz H. (1988) Mammalian lignans and phyto-oestrogens. Recent studies on their formation, metabolism, and biological role in health and disease. In: Rowland IA., ed. The Role of Gut Microflora in Toxicity and Cancer. New York: Academic Press, pp. 315–345. Shutt DA. Cox RI. (1972) Steroid and phytoestrogen binding to sheep uterine receptors in vitro. J Endocr. 52, 299–307. Singh AK. (2007) Screening and Fate of Estrogens in Pig Excreta and Manure Samples. Research report, National Pork Board, Des Moines, IA, USA. Sirtori CR. Arnoldi A. Johnson SK. (2005) Phytoestrogens: End of a tale? Ann Med. 37, 423–438. Soucy NV. Parkinson HD. Sochaski MA. Borghoff SJ. (2006) Kinetics of genistein and its conjugated metabolites in pregnant Sprague-Dawley rats following single and repeated genistein administration. Toxicol Sci. 90, 230–240. Stitch SR. Toumba JK. Groen MB. Funke CW. Leemhuis J. Vink J. Woods GF. (1980) Excretion, isolation, and structure of a new phenolic constituent of female urine. Nature. 287, 738–740. Valentin-Blasini L. Blount BC. Rogers HS. Needham LL. (2000) HPLC-MS/MS method for the

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measurement of seven phytoestrogens in human serum and urine. J Exposure Anal Environ Epid. 10, 799–807. Walle T. (2004) Flavonoids and isoflavones (phytoestrogens): Absorption, metabolism, and bioactivity. Free Radical Biol Med. 36, 829–837. Ward RS. (1995) Lignans, neolignans, and related compounds. Nat Prod Rep. 12, 183–205. Ward RS. (1993) Lignans, neolignans, and related compounds. Nat Prod Rep. 10, 1–28. Whitten PL. Kudo S. Okubo KK. (1997) Isoflavonoids. In: D’Mello JPF., ed. Handbook of Plant and Fungal Toxicants, Boca Raton, FL: CRC Press, pp. 117–137.

Xu X. Harris KS. Wang H-J. Murphy PA. Hendrich S. (1995) Bioavailability of soybean isoflavones depends upon gut microflora in women. J Nutr. 125, 2307– 2315. Zhang Y. Wang G-J. Song TT. Murphy PA. Hendrich S. (1999) Urinary disposition of the soybean isoflavones daidzein, genistein and glycitein differs among humans with moderate fecal isoflavone degradation activity. J Nutr. 129, 957–962. Zuo YB. Zeng AW. Yuan XG. Yu KT. (2008) Extraction of soybean isoflavones from soybean meal with aqueous methanol modified supercritical carbon dioxide. J Food Eng. 89, 384–389.

Chapter 8 Mycoestrogens Jean-Denis Bailly

Introduction Endocrine-disrupting compounds have received a lot of public attention for many years because they are suspected to reduce male fertility in human and wildlife populations and possibly to be involved in the development of several cancers (Stopper et al. 2005). Among these compounds, one molecule, zearalenone, is a secondary metabolite produced by fungi that are frequent contaminants of cereal grains. This compound is therefore able to enter, directly or indirectly, both the human and animal food chain. Zearalenone is well known by farmers because this mycotoxin is often responsible for reproduction disorders, especially in pigs. Therefore, the content of this natural contaminant is regulated in many foodstuffs, and many analytical methods were developed to quantify the contamination level in many foods from both vegetal and animal origin.

Origin of zearalenone Zearalenone has been isolated for the first time from maize contaminated with Gibberella zeae, the anamorph of Fusarium graminearum (Stob et al. 1962). This mycotoxin can be produced by several Fusarium species such as F. graminearum, F. prolifera-

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

tum, F. culmorum, and F. oxysporum (Molto et al. 1997; Sydenham et al. 1991). These fungal species usually develop on living plants, and zearalenone contamination occurs in the field, during periharvest, or in early storage, when the drying step was not sufficient. Indeed, Fusarium growth and mycotoxin production require high water activity (>0.90) (Montani et al. 1988; Jimenez et al. 1996). The temperature for zearalenone production is lower than the optimal temperature for mycelium development and is about 20– 25°C (Llorens et al. 2004; Ryu and Bullerman 1999; Milano and Lopez 1991). It has also been noted that successive variations of ambient temperature could increase zearalenone production. This can be encountered in traditional cribs used to dry and store maize cobs. Zearalenone production is also favored in substrates with high glucid/protein ratio. Due to all these parameters, zearalenone appears to be a frequent contaminant of cereals and cereal-derived products in European and other countries with temperate climates (Schothorst and van Egmond 2004; Zinedine et al. 2007). It is more frequently found in maize and wheat grains. Zearalenone contamination has also been reported in hay and badly dried straw (Scudamore and Livesey 1998). Silage can also be contaminated with zearalenone. Indeed, in the case of insufficient packing, weak acidification in the first hours, or slow progression at the front of the silo, the Fusarium species may develop and produce zearalenone (Bailly et al. 2006). 229

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Analysis of Endocrine Disrupting Compounds in Food

Toxicological features Acute toxicity of zearalenone is usually considered to be weak, with LD50 after oral ingestion ranging from 2000 to more than 20,000 mg/kg b.w. (Kuiper-Goodman et al. 1987; JECFA 2000). Subacute and chronic toxicity of the mycotoxin is more frequent and may be observed at the natural contamination levels of feeds. Strong variations of sensitivity are usually observed among animal species, and differences in the metabolism of the mycotoxin after ingestion could be responsible for these observations.

Metabolism As for many other mycotoxins, metabolism of zearalenone after ingestion in food or feed is key to our understanding of the molecule’s toxicity. Zearalenone is quickly absorbed after oral ingestion (Dailey et al. 1980; Olsen et al. 1985). Urinary excretion of zearalenone and its metabolites suggest that the absorption rate is high (Kuiper-Goodman et al. 1987; Mirocha et al. 1981) and was estimated to be 80–85% in pigs after a single oral dose (Biehl et al. 1993). Zearalenone can be metabolized in digestive tracts by both microflora and intestinal mucosa (Kollarczik et al. 1994; Olsen et al. 1987). This metabolism results in the appearance of α− and β-zearalenol and α− and β-zearalanol. The proportion of these two metabolites may change depending on the animal species (Zinedine et al. 2007; Olsen et al. 1987; Kallela and Vasenius, 1982). After absorption, two major hepatic biotransformation pathways may occur for zearalenone in animals (Olsen et al. 1981; Kiessling and Pettersson 1978): • hydroxylation resulting in the formation of α-zearalenol and β-zearalenol • conjugation of zearalenone and reduced metabolites with glucuronic acid Because among zearalenone metabolites α-zearalenol has a higher affinity for estrogenic receptors, its appearance throughout

the metabolic pathways can be considered as a bioactivation. Therefore, metabolism of zearalenone and differences in toxin transformation within an organism can explain differences in toxicity observed in several animal species (Gaumy et al. 2001a). Differences between species in hepatic biotransformation have been demonstrated; pigs seem to convert zearalenone predominantly into α-zearalenol, whereas β-zearalenol is the main metabolite in cattle (Malekinejad et al. 2006). In humans, as in pigs, zearalenone was found mainly as glucoronide conjugates of zearalenone and α-zearalenol in urine. All of the metabolites found in humans during the 24 h of sampling were glucuronides (Mirocha et al. 1981). Zearalenone and its metabolites are excreted in urine or bile (JECFA 2000). In ruminants, zearalenone and its metabolites are detected in bile at the rates of 68% βzearalenol, 24% α-zearalenol, and 8% zearalenone (Danicke et al. 2002a). In this study, neither zearalenone nor its metabolites were detected in muscles, kidney, liver, or dorsal fat of bovine receiving 0.1 mg zearalenone/day/ kg feed. In other species, few studies are available on the potential carryover of this mycotoxin in animal edible organs. It appears that, at least in pigs, meat and other edible parts may not be contaminated, even after exposure of the animals to high concentrations of the toxin (Sundlof and Strickland 1986; Baldwin et al. 1983; Goyarts et al. 2007). In poultry, few studies done with very high doses of zearalenone allowed the detection of the toxin at a detectable level in muscles (Mirocha et al. 1982). More recently, an experiment of long-term exposure of laying hens with 1.58 mg zearalenone/kg feed for 16 weeks did not allow the detection of any residues in muscles, fat, or eggs (Danicke et al. 2002b).

Toxicity The main effects of zearalenone are directly related to the fixation of ZEA and its metabolites on estrogenic receptors (Takemura et al. 2007) leading to acute and subacute repro-

Mycoestrogens

ductive disorders. Several reviews were devoted to zearalenone toxicity, and only the main features will be reported here (Zinedine et al. 2007; Minervini and Dell’Aquila 2008; AFSSA 2009) Reproductive disorders in animals In vitro and in vivo experiments demonstrated that zearalenone and its metabolites are able to competitively bind to estrogen receptors present in several tissues, such as uterus, mammary glands, liver, and hypothalamus. Affinity with estrogenic receptors are, in decreasing order, α-zearalanol > αzearalenol > β-zearalanol > zearalenone > βzearalenol (Kuiper-Goodman et al. 1987). The disorders that will follow zearalenone ingestion may vary depending on the animal species and the metabolism of the native toxin (α- vs. β-zearalenol production). Pig and sheep appear more sensitive than other animal species: in multiple exposure experiments, the no observed effect level (NOEL) in pigs was 40 μg/kg of body weight, whereas it was 100 μg/kg b.w. in rats (KuiperGoodman et al. 1987; JECFA 2000). The specific symptoms are related to the concentration of the mycotoxin and the stage of estrus cycle or pregnancy. In young gilt, exposure for several days to a feed containing 1 to 5 mg zearalenone/kg leads to clinical signs such as hyperemia, edematous swelling of the vulva, and sometimes vaginal and even rectal prolapse (Minervini and Dell’Aquila 2008). Many other impacts on reproductive organs were reported. Zearalenone induces alteration in the reproductive tracts of both laboratory and farm animals. This mycotoxin also leads to a decrease of fertility; decrease in litter size; an increase in embryo-lethal resorptions; and change in adrenal, thyroid, and pituitary gland weights of exposed animals. In male pigs, zearalenone can depress testosterone, weight of testes, and spermatogenesis while inducing feminization and suppressing libido (Kuiper-Goodman

231

et al. 1987; JECFA 2000; Zinedine et al. 2007). Genotoxicity and carcinogenicity The International Agency for Research on Cancer (IARC) established in 1993 that SOS-chromotest of DNA reparation done on Bacillus subtilis was negative, whereas the results of the rec-assay on this same bacteria was positive. Zearalenone does not induce genetic modification on Salmonella typhimurium (Ames test) or genetic recombination on S. cerevisiae (IARC 1993); however, the molecule leads to sister chromatid exchanges in Chinese hamster ovary cells and to DNA adducts in the liver and kidney of exposed mice (Pfohl-Leszkowicz et al. 1995; Grosse et al. 1997). In vivo, the percentage of chromosomal aberration increases in the bone marrow of mice receiving 10 to 40 mg zearalenone/kg b.w. (Ouanes et al. 2005). Concerning carcinogenicity, zearalenone was evaluated by IARC and classified in group III of molecules for which animal evidence of carcinogenicity was not sufficient. Until now, long-term experiments done in rodents did not demonstrate a carcinogenic potential of the molecule. Toxicity in humans In humans, zearalenone (or zearalanol) was suspected to be responsible for numerous cases of early puberty in young children in Puerto Rico between 1978 and 1981 (Saenz de Rodriguez 1984; Saenz de Rodriguez et al. 1985). Several studies did not allow a conclusion that zearalenone was responsible for such modification, and another hypothesis involving phthalates was proposed (Colon et al. 2000; Larriuz-Serrano et al. 2001).

Regulation In 1999, the Joint Expert Committee on Food Additives (JECFA) established a temporary maximal daily tolerable dose of 0.5 μg/kg

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Analysis of Endocrine Disrupting Compounds in Food

Table 8.1. EU regulation concerning zearalenone in foods and feeds. Food

Feeda

a

Nature

Maximal Value (ng/g)

Reference

Cereals (except maize) Maize Cereal flour (except maize flour) Maize flour Bread, biscuits, pastries Breakfast cereals Maize-based baby food Cereal-based baby food Cereals and cereal-based products (except maize) Maize by-products Complementary feed for • piglets and gilts • sows and bacon pigs • calves, lactating cattle, ovines, caprines

100 200 75 200 50 50 20 20 2000 3000

EU, L143/7, 2005

EU, L229/9, 2006

100 250 500

Recommendations. CH3

O

HO

O

OH

O

Figure 8.1. Structure of zearalenone.

body weight. It is based on the hormonal effects observed in the most sensitive species (pigs) and the NOEL of 50 μg/kg b.w./day, with a security factor of 100 (JECFA 2000). In France, the Conseil Superieur d’Hygiène Publique de France (CSPHF) proposed a daily tolerable dose of 0.1 μg/kg b.w./day, calculated on effects observed on monkey reproduction (CSHPF 1999). In 2003, ZEA was regulated in foods and feeds by 16 countries, and in 2005 and 2006, the European Union adopted regulations and recommendations for zearalenone in human foods and animal feeds (European Union 2005, 2006) (Table 8.1).

Zearalenone analysis Physicochemical properties The structure of zearalenone is shown in Figure 8.1. The natural metabolites of the native toxin, α- and β-zearalenol, correspond to the reduction of the ketone function in C6.

Zearalenone has a molecular weight of 318 g/mol. This compound is weakly soluble in water and in hexane. Its solubility increases with polarity of solvents: benzene, chloroform, ethyl acetate, acetonitrile, acetone, methanol, ethanol (Hidy et al. 1977). Acetonitrile is commonly used for its extraction in foods and feeds. The molecule has three maximal absorption wavelengths in UV: 236, 274, and 314 nm. The 274-nm peak is the most characteristic and commonly used for UV detection of the toxin. Zearalenone emits a blue fluorescence with maximal emission at 450 nm after excitation between 230 and 340 nm in ethanol (Gaumy et al. 2001b).

Methods of analysis Due to regulatory limits, methods for analysis for zearalenone content in foods and feeds may allow the detection of several nanograms per gram. Reviews have been published to detail analytical methods available (Krska 1998; Ksrka and Josephs 2001; Zöllner and Mayer 2006). It has to be noted that zearalenone is sensitive to light exposure, especially when in solution. Therefore, preventive measures have to be taken to avoid this photodegradation.

Mycoestrogens

Extraction Solvents used for liquid extraction of zearalenone and its metabolites are mainly ethyl-acetate, methanol, acetonitrile, and chloroform, alone or mixed. The mixture of acetonitrile and water is the most commonly used. For solid matrices, more sophisticated and efficient methods may be applied, for example, ultrasound or microwaves (Pallaroni et al. 2002; Pallaroni and von Holst 2003). In biological matrixes (plasma, urine, feces), a step of hydrolysis of phase II metabolites is necessary before the purification procedure, which can be achieved by enzymatic or chemical protocol (Zöllner et al. 2002). In vegetal materials, the demonstrated presence of sulfate-conjugates (Plasencia and Mirocha 1991) or glucoside-conjugates (Gareis et al. 1990) is rarely taken into account in routine methods. The real contamination with zearalenone may be underevaluated.

Purification Purification of zearalenone may be achieved using liquid–liquid extraction (LLE), solidphase extraction (SPE), or immunoaffinity (IAC) procedures. For SPE, most stationary phases may be used: inverse phase (C18, C8, or C4), normal phase (Florisil, SiOH, NH2), or anion exchange (SAX) (Llorens et al. 2002). Another approach using the ready-touse column Mycosep (Romer Labs Inc.) may be applied. These columns are adsorbants (charcoal, celite, ion exchange resin) mixed in a plastic tube. It allows a rapid purification of sample without any rinsing and with a selective retention of impurities (Silva and Vargas 2001). IAC columns have also been developed for zearalenone and are very popular (De Saeger et al. 2003; Eskola et al. 2002; Fazekas and Tar 2001; Kruger et al. 1999; Zöllner et al. 1999; Visconti and Pascale 1998; Rosenberg et al. 1998). Although purification is very selective and extraction yields are usually high, it has

233

to be noted that the antibody may not have the same affinity for all metabolites, resulting in some not being accurately extracted. Fixation capacity of columns is limited, and a great number of interfering substances may perturb the purification by saturation of the fixation sites (Llorens et al. 2002). Finally, the possible re-use of these columns raises the risk of cross-contamination of samples. Quantification As for many mycotoxins, thin layer chromatography (TLC) was widely used to quantify zearalenone, using the fluorescence of the molecule. One method has been adopted by AOAC International (Scott 1995) and TLC is still used in some surveys done on zearalenone contamination of cereals (Gonzalez Pereyra et al. 2008; Oliveira et al. 2006). Indeed, detection limit of tens of micrograms per kilogram is compatible with existing regulation for animal feed (Quiroga et al. 1994; Dawlatana et al. 1998; De Oliveira and Flavio 1998) (Table 8.2). High-performance liquid chromatography (HPLC) is more widely used for zearalenone quantification in cereals. Many methods using C18 as a stationary phase and CH3CN/ H2O as a mobile phase have been described. More specific stationary phases have also been proposed, such as molecular printing (Weiss et al. 2003). Detectors are often fluorimeters (De Saeger et al. 2003; Eskola et al. 2002; Fazekas and Tar 2001; Kruger et al. 1999; Ware et al. 1999; Visconti and Pascale 1998) or UV detectors (Llorens et al. 2002; Fazekas et al. 2001). Sensitivity of these methods varies depending on the metabolites and is less important for reduced metabolites (α- and β-zearalenol). The most characteristic methods are reported in Table 8.3. More recently, a methodology for multimycotoxin analysis, among which methodologies for zearalenone; aflatoxin B1, B2, G1, G2; ochratoxin A; deoxynivalenol; and fumonisin B1 and B2 were developed for corn analysis.

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Analysis of Endocrine Disrupting Compounds in Food

Table 8.2. Zearalenone quantification in foods by TLC methods. Food

Extraction

Cleanup

Quantification

DL (μg/kg)

Reference

Corn

Water-acetonitrile (1:5)—diatomaceous earth

Silica gel column chromatography and liquid– liquid partition

<300

Scott 1995

Corn

Acetonitrile—4% KCl (90:10)

Precipitation and liquid–liquid partition

50

Quiroga et al. 1994

Corn flour

Water-acetonitrile (1:5)—diatomaceous earth

Silica gel column chromatography and liquid– liquid partition

100

De Oliveira and Flavio 1998

Pig feed

Methanol/water (60:40)

Liquid–liquid partition

TLC separation in chloroform-alcohol Derivatization with aluminium chloride (20%) and heating 5 min at 130°C TLC separation in chloroform (1% ethanol)/acetone (90:10) Visualization of fluorescence under UV light (254 nm) TLC separation in benzene/hexane (3:1) then toluene/ ethylacetate/formic acid (60:30:15) Derivatization with bis-diazotized benzidine TLC separation in chloroform/acetone (90:10) Visualization of fluorescence under UV light (254 nm)

100

GonzalezPereyra et al. 2008

TLC, thin-layer chromatography.

This method uses immunoaffinity, solidphase extraction columns, and HPLC with postcolumn derivatization (Ofitserova et al. 2009). For zearalenone, it displayed limits of detection of 168 μg/kg. Due to metabolism of the native molecule and the very weak carry over of zearalenone in edible parts of farm animals (see Metabolism), few classical HPLC-ultra violet or HPLC-fluorescence techniques were developed for zearalenone detection in the edible parts of farm animals (Medina and Sherman 1986; Curtui et al. 2001; Danicke et al. 2002; Danicke et al. 2007; Goyarts et al. 2007). Zearalenone and its metabolites can also be detected by gas chromatography (GC). However, the usefulness of this method is limited due to the time-consuming need for derivatization of phenolic hydroxy groups. Consequently, only GC/mass spectrometry

(MS) has been applied to confirm positive results (Tanaka et al. 2000; Ryu et al. 1996). In recent years, many LC/MS detection methods have also been proposed for zearalenone and its metabolites (for reviews see Zöllner and Mayer-Helm 2006; Krska et al, 2007). The high selectivity of MS compared to other detection techniques allows simplification of cleanup procedures, even for complex matrices such as compound feeds and animal tissues. The method of chemical ionization at atmospheric pressure is the most commonly used, followed by electrospray (Pallaroni et al. 2002; Jodlbauer et al. 2000; Zöllner et al. 2000; Kleinova et al. 2002; Rosenberg et al. 1998). These methods allow the detection of zearalenone and its metabolites at levels below 1 ng/g (Zöllner and Mayer-Helm 2006) and are now commonly used in many laboratories specializing in human or animal feed analysis.

Table 8.3. Zearalenone quantification by HPLC. Food

Extraction

Cleanup

Cereals

Acetonitrile/water (3:1)

Florisil column

Cereals

Acetonitrile/4% KCl (90:10)

Liquid–liquid partition

Cereals

Acetonitrile/water (90:10)

Immunoaffinity column

Corn

Acetonitrile/4% KCl (90:10)

Liquid–liquid partition and Florisil column

Corn

Dichloromethane/ water (10:1)

Liquid–liquid partition

Corn

Acetonitrile/water (90:10)

Immunoaffinity column

Corn

Methanol/water (85:15) α-zearalanol as internal standard

Florisil column and liquid–liquid partition

Cornbased food

Acetonitrile/water (75:25)

Immunoaffinity column

Quantification HPLC Separation on silica gel, mobile phase 90% water-saturated chloroform/ cyclohexane/ acetonitrile/ethanol (50:15:2:1) Fluorescence detection at 460 nm (ex: 176 nm) HPLC Separation on reverse phase C18, mobile phase methanol/water (80:20) Fluorescence detection at 440 nm (ex: 285 nm) HPLC Separation on reverse phase C18, mobile phase water/ acetonitrile/methanol (43:35:22) Fluorescence detection at 440 nm (ex: 274 nm) HPLC Separation on reverse phase C18, mobile phase methanol/water (70:30) Fluorescence detection at 418 nm (ex: 236 nm) HPLC Separation on reverse phase C18, mobile phase methanol/water (80:20) Fluorescence detection at 465 nm (ex: 280 nm) HPLC Mobile phase acetonitrile/water/ methanol (46:46:8) Fluorescence detection at 440 nm (ex: 274 nm) HPLC Separation on reverse phase C18, mobile phase acetonitrile/ water/methanol (50:42:8) UV detection at 236, 274 and 316 nm HPLC Separation on reverse phase C18, mobile phase methanol/water (70:30) Fluorescence detection at 450 nm (ex: 274 nm)

DL (μg/kg) 2.0

Reference Tanaka et al. 1985

Hetmanski and Scudamore 1991

0.25

Thongrussamee et al. 2008

2.0

Kang et al. 1994

1.5–2.2

Chang and De Vries 1984

3.0

Visconti and Pascale 1998

0.7

Briones-Reyes et al. 2007

Nuryono et al. 2005

(continued)

235

Table 8.3. Zearalenone quantification by HPLC. (cont.) Food

Extraction

Cleanup

Quantification HPLC Separation on reverse phase C18, mobile phase acetonitrile/0.3% sodium acetate (11:10) Fluorescence detection at 446 nm (ex: 274 nm) HPLC Separation on ODS Hypersil column, mobile phase acetonitrile/water (40:60) Fluorescence detection at 450 nm (ex: 275 nm) HPLC Separation on reverse phase C18, mobile phase acetonitrile/ water (50:50) Fluorescence detection at 440 nm (ex: 274 nm) HPLC Separation on reverse phase C18 Fluorescence detection at 450 nm (ex: 235 nm) HPLC Separation on Partisil column, mobile phase isooctane/chloroform/ methanol (35:25:3) UV detection at 280 nm HPLC Separation on ODS Hypersil column, mobile phase acetonitrile/methanol (50:50) Fluorescence detection at 440 nm (ex: 274 nm) HPLC Reverse phase compression system, mobile phase acetonitrile/water (60:40) UV detection at 280 nm HPLC Separation on reverse phase C18, mobile phase methanol/water/ acetonitrile (61:35:4) Fluorescence detection at 470 nm (ex: 236 nm)

Barley

Methanol/water (60:40) Zearalenone 6′oxime as internal standard

Liquid–liquid partition and piperidinohydroxypropylsephadex LH-20 column

Poultry feed

Acetonitrile/water (75:25)

Immunoaffinity column

Animal feed

Acetonitrile/water (90:10)

Immunoaffinity column

Food of plant origin

Acetonitrile/water (90:10)

Immunoaffinity column

Rat liver

Methylene chloride

Liquid–liquid partition and Sephadex LH-20 resin

Rat tissues

Methanol and treatment with b-glucoronidase solution (5000 U/mL) for 16 h at 37°C.

Immunoaffinity column

Chicken tissues

Acetonitrile

Liquid–liquid extraction

Milk

Basification with ammonium hydroxide Acetonitrile

Liquid–liquid extraction and aminopropyl SPE column

HPLC, high-performance liquid chromatography.

236

DL (μg/kg)

Reference

1.0

Tanaka et al. 1993

7.0

Labuda et al. 2005

De Saeger et al. 2003

1.0

Schollenberger et al. 2005

2.0

James et al. 1982

1.3

Bernhoft et al. 2001

Turner et al. 1983

0.2–2.0

Scott and Lawrence 1988

237

ZEA, α-zearalenol, zeranol

Bovine muscle

Urine, plasma, feces of horses

ZEA, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol

Fish liver and tissues

IAC

Enzymatic deglucoronidation/ desulfation, SPE with RP-18 Enzymatic deglucoronidation/ desulfation, SPE with RP-18, IAC

SPE with carbograph-4 columns

Bovine hair

Urine

SPE with RP-18 column

Liver, urine, and muscle of pigs

ZEA, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanolzeranol ZEA, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol ZEA, α-zearalenol, β-zearalenol,

ZEA, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol

SPE with RP-18 columns

Pig tissues

SPE with RP-18 columns

Sample Cleanup

ZEA, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol

Matrix

Hp-5 ms capillary

Column

RP-18

RP-18

RP-18

RP-18

RP-18

RP-18

Column

LC

Helium

Carrier gas

H2O/MeOH/ACN (35:30:35)

ESI-APCI

H2O/MeOH/ACN (47:16:37) with 10 mM NH4OAc H2O/MeOH (16:84)

ACPI

Ionization

ACPI

ESI

APCI Positive ion mode ACPI Positive ion mode ESI

Ionization

H2O/MeOH/ACN (45:45:10) with 15 mM NH4AOc H2O/MeOH/ACN (45:45:10) with 15 mM NH4AOc Gradient H2O/ ACN

Mobile phase

MS

Scan mode SIM

SIM

SRM

SRM

SRM

SRM

Scan Mode SRM

1 ng/g

0.5–1

1

0.1–1

0.5–1 (muscle) 0.1–1 (liver) 1.6

0.5–1

LOQ (ng/g)

Zhang et al. 2006

Songsermsakul et al. 2006

Launay et al. 2004

Lagana et al. 2003, 2004

Hooijering et al. 2005

Zöllner et al. 2002

Kleinova et al. 2002

Reference

ACN, acetonitrile; ACPI, atmospheric pressure chemical ionization; API, atmospheric pressure ionization; ESI, electron spray ionization; IAC, immuno-affinity column; LOQ, limit of quantification; MeOH, methanol; SIM, selected ion monitoring; SPE, solid-phase extraction; SRM, selected reaction monitoring; ZEA, zearalenone.

GCMS

LCMS

Analytes

Table 8.4. Overview of LC-MS and GC-MS methods for zearalenone analysis in animal tissues.

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Analysis of Endocrine Disrupting Compounds in Food

The developments of LC/MS and GC/MS also allowed an improvement of sensitivity in zearalenone and metabolite detection in animal tissues (Table 8.4). It also makes possible the discrimination between the use of estrogen as growth promoters during breeding (Zeranol) and the exposure of animals to a contaminated feed (Launay et al. 2004; Blokland et al. 2006; Xia et al. 2009). The other major interest in LC/MS methods is the possibility of multidetection of several mycotoxins belonging to different families, such as aflatoxins, trichothecenes, fumonisins, ochratoxin A, and zearalenone with good detection limits of few micrograms per kilogram. (Di Mavungu et al. 2009). Over last few years, immunoanalytical methods such as ELISA became very popular as rapid screening tools for mycotoxin contamination of cereals. The main advantages of such techniques are that they do not require expansive apparatus, they are easy to use, and they require only minimal or no cleanup procedure. For quantification of zearalenone and its metabolites in cereals and other matrices, several immunological methods have also been set up, including RIA and ELISA (Meyer et al. 2002; Pichler et al. 1998; Josephs et al. 2001; Lee et al. 2001; Bennett et al. 1994). Many ELISA kits are commercially available. The limit of quantification of these methods is about several tens of nanograms per gram, and one has been validated by AOAC as a rapid method (AOAC 994.01) (Association of Analytical Chemistry 2002). The ELISA method reliability may depend on the matrix and lead to overestimation of contamination level (Josephs et al. 2001). The kits also show a cross-reactivity with α- and β-zearalenol (Maragos and Kim 2004). The development of rapid and reliable methods for screening of cereal batches in the field is of great interest. One of these methodologies is based on the use of monoclonal antibodies fixed on membranes instead of microwells (Burmistrova et al. 2009). Results are usually qualitative or

semiquantitative. A test for simultaneously analyzing zearalenone and ochratoxin A was recently developed (Shim et al. 2009).

Conclusion Zearalenone is a mycotoxin with estrogenic activity that leads to reproductive disorders in animals and may also have importance in human health. Many analytical methods were developed to control zearalenone content in foods and feeds and levels of detection of these methods are highly sufficient compared to existing regulations for both human and animal nutrition. The remaining problem is the existence of bound derivative that may not be quantified using classical extracting procedures, which leads to underestimation of the real level of contamination. These masked mycotoxins are receiving particular attention, and it is likely that new extraction procedures will soon be developed to solve that problem. Note also that if zearalenone is the only mycotoxin with real estrogenic activity in humans and animals, fungal development on plants may also contribute to accumulation of other components with estrogenic activity: the phytoestrogens, and particularly coumestrol, which is frequently involved in reproductive disorders in small ruminants (see Chapter 7, Phytoestrogens).

References Agence Française de Sécurité Sanitaire des Aliments (AFSSA). 2009. Evaluation des risques liés à la présence de mycotoxines dans les chaînes alimentaires humaines et animales. AFSSA ed, Paris. 339 pp. Available from www.afssa.fr. Association of Analytical Chemistry (AOAC). 2002. Official method 994.01, zearalenone in corn, wheat and feed, enzyme linked immunosorbent (agri-screen) method. J AOAC Int. Association of Analytical Chemistry (AOAC). 2002. Official method 994.01, zearalenone in corn, wheat and feed, enzyme linked immunosorbent (agri-screen) method. J AOAC Int. Bailly, J.D., Bailly, S., Le Bars, J. 2006. Les alterations fongiques de l’ensilage de maïs: Consequences et conduite à tenir. Bull GTV. 7, 39–43.

Mycoestrogens

Baldwin, R.S., Williams, R.D., Terry, M.K. 1983. Zeranol: A review of the metabolism, toxicology, and analytical methods for detection of tissue residues. Regul Toxicol Pharmacol. 3, 9–25. Bennett, G.A., Nelsen, T.C., Miller, B.M. 1994. Enzymelinked immunosorbent assay for detection of zearalenone in corn, wheat, and pig feed: Collaborative study. J AOAC Int. 77, 1500–1509. Bernhoft, A., Behrens, G.H.D., Ingebritsen, K., Langseth, W., Berndt, S., Haugen, T.B., Grotmol, T. 2001. Placental transfer of the estrogenic mycotoxin zearalenone in rats. Reprod Toxicol. 15, 545–550. Biehl, M.L., Prelusky, D.B., Koritz, G.D., Hartin, K.E., Buck, W.B., Trenholm, H.L. 1993. Biliary excretion and enterohepatic cycling of zearalenone in immature pigs. Toxicol Appl Pharmacol. 121, 152–159. Blokland, M.H., Sterk, S.S., Stephany, R.W., Launay, F.M., Kennedy, D.G., van Ginkel, L.A. 2006. Determination of resorcyclic acid lactones in biological samples by GC-MS. Discrimination between illegal use and contamination with Fusarium toxins. Anal Bioanal Chem. 384, 1221–1227. Briones-Reyes, D., Gomez-Martinez, L., Cueva-Rolon, R. 2007. Zearalenone contamination in corn for human consumption in the state of Tlaxcala, Mexico. Food Chem. 100, 693–698. Burmistrova, N.A., Goryacheva, I.Y., Basova, E.Y., Franki, A.S., Elewaut, D., Van Beneden, K., Deforce, D., Van Peteghem, C., De Saeger, S. 2009. Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats. Anal Bioanal Chem. 395, 1301–1307. Chang, H.L. and De Vries, J.W. 1984. Short liquid chromatographic method for determination of zearalenone and alpha zearalenol. J Assoc Off Anal Chem. 67, 741–744. Colon, I., Caro, D., Bourdony, C.J., Rosario, O. 2000. Identification of phtalate esters in the serum of young Puerto Rican girls with premature breast development. Environ. Health Perspect. 108, 895–900. Conseil Superieur d’Hygiène Publique de France (CSPHF). 1999. Les mycotoxines dans l’alimentation: évaluation et gestion du risque. Chap 18. Conclusion et recommendations, p 451–456. Tec&Doc ed., Paris. Curtui, V.G., Gareis, M., Usleber, E., Martlbauer, E. 2001. Survey of Romanian slaughtered pigs for the occurrence of mycotoxins oxhratoxins A and B, and zearalenone. Food Addit Contam. 18, 730–738. Dailey, R.E., Reese, R.E., Brouwer, E.A. 1980. Metabolism of (14C)zearalenone in laying hens. J Agric Food Chem. 28, 286–291. Danicke, S., Gadeken, D., Uberschar, K.H., Meyer, U., Scholtz, H. 2002a. Effects of Fusarium toxin contamined wheat and of a detoxifying agent on performance of growing bulls, on nutrient digestibility in wethers and on the carry over of zearalenone. Arch Tierernahr. 56, 245–261. Danicke, S., Uberschar, K.H., Halle, I., Matthes, S., Valenta, H., Flachowsky, G. 2002b. Effect of addition of a detoxifying agent to laying hen diets containing uncontaminated or Fusarium toxin-contamined maize on performance of hens and on carryover of zearalenone. Poult Sci. 81, 1671–1680.

239

Danicke, S., Valenta, H., Ueberschar, K.H., Matthes, S. 2007. On the interactions between Fusarium toxincontamined wheat and non-starch-polysaccharide hydrolysing enzymes in turkey diets on performance, health, and carry-over of deoxynivalenol and zearalenone. Br Poult Sci. 48, 39–48. Dawlatana, M., Coker, R.D., Nagler, M.J., Blunden, G., Oliver, G.W.O. 1998. An HPTLC method for the quantitative determination of zearalenone in maize. Chromatographia. 47, 215–218. De Oliveira, S.C. and Flavio, M.A. 1998. A simplified method for the determination of zearalenone in cornflour. Alimentaria. 298, 27–29. De Saeger, S., Sibanda, L., Van Peteghem, C. 2003. Analysis of zearalenone and α-zearalenol in animal feed using high-performance liquid chromatography. Anal Chim Acta. 487, 137–143. Di Mavungu, J.D., Monbaliu, S., Scippo, ML., MaghuinRogister, G., Schneider, Y.J., Larondelle, Y., Callebaut, A., Robbens, J., Van Peteghem, C., De Saeger, S. 2009. LC-MS/MS multi-analyte method for mycotoxin determination in food supplements. Food Addit Contam. 26, 885–895. Eskola, M., Kokkonen, M., Rizzo A. 2002. Application of manual and automated systems for purification of ochratoxinA and zearalenone in cereals with immunoaffinity columns. J Agric Food Chem. 50, 41–47. European Union. 2005. Règlement no. 856/2005, Les toxines du Fusarium, Off J Europ Un. L 143, 3. European Union. 2006. Recommandation (576/2005): La présence de déoxynivalénol, de zéaralénone, d’ochratoxine A, des toxines T-2 et HT-2 et des fumonisines dans les produits destinés à l’alimentation animale. Off J Europ Un. L 229, 7. Fazekas, B. and Tar, A. 2001. Determination of zearalenone content in cereals and feedstuffs by immunoaffinity column coupled with liquid chromatography. J AOAC Int. 5, 1453–1459. Gareis, M., Bauer, J., Thiem, J., Plank, G., Grabley, S., Gedek, B. 1990. Cleavage of zearalenone-glycoside, a “masked” mycotoxin, during digestion in swine. J Med B. 37, 236–240. Gaumy, J.L., Bailly, J.D., Bénard, G., Guerre, P. 2001. Zearalénone: Origine et effets chez les animaux d’élevage. Rev Med Vét. 152, 123–136. Gaumy, J.L., Bailly, J.D., Bénard, G., Guerre, G. 2001. Zearalénone: Propriétés et toxicité expérimentale. Rev Med Vét. 152, 219–234. Gonzalez-Pereyra, M.L., Pereyra, C.M., Ramirez, M.L., Rosa, C.A., Dalcero, A.M., Cavaglieri, L.R. 2008. Determination of mycobiota and mycotoxins in pig feed in central Argentina. Lett Appl Microbiol. 46, 555–561. Goyarts, T., Danicke, S., Valenta, H., Ueberschar, K.H. 2007. Carry-over of Fusarium toxins (deoxynivalenol and zearalenone) from naturally contamined wheat to pigs. Food Addit Contam. 24, 369–380. Grosse Y., Chekir-Ghedira L., Huc A., Obrecht-Pflumio S., Dirheimer G., Bacha H., Pfohl-Leszkowicz A. 1997. Retinol, ascorbic acid and alpha tocopherol prevent DNA adduct formation in mice treated with the mycotoxins ochratoxin A and zearalenone. Cancer Lett. 19, 225–229.

240

Analysis of Endocrine Disrupting Compounds in Food

Hetmanski, M.T. and Scudamore, K.A. 1991. Detection of zearalenone in cereal extracts using high performance liquid chromatography with post-column derivatization. J Chromatogr. 588, 47–52. Hidy, P.H., Baldwin, R.S., Greasham, R.L., Keith, C.L., McMullen, J.R. 1977. Zearalenone and some derivates: Production and biological activities. Adv Appl Microbiol. 22, 59–82. Hooijering, H., Lommen, A., Mulder, P.P.J., van Rhijn, J.A., Nielen, M.W.F. 2005. Liquid chromatographyelectrospray ionisation mass spectrometry based method for the determination of estradiol benzoate in hair of cattle. Anal Chim Acta. 529, 167–172. International Agency for Research on Cancer (IARC). 1993. Some naturally occurring substances, food items and constituents, heterocyclic aromatic amines and mycotoxins, in Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, France: World Health Organization (WHO). 56, 397–444. James, L.J., McGirr, L.G., Smith, T.K. 1982. High pressure liquid chromatography of zearalenone and zearalenols in rat urine and liver. J Assoc Anal Chem. 65, 8–13 Joint Expert Committee on Food Additives (JECFA). 2000. Safety evaluation of certain food additives. World Health Organization (WHO), Food Additives Series 44, 53rd Report. Jimenez, M., Manez, M., Hernandez, E. 1996. Influence of water activity and temperature on the production of zearalenone in corn by three Fusarium species. Int J Food Microbiol. 29, 417–421. Jodlbauer, J., Zöllner, P., Lindner, W. 2000. Determination of zeranol, taleranol, zearalenone, α- and β-zearalenol in urine and tissue by high-performance liquid chromatography-tandem mass spectrometry. Chromatographia. 51, 681–687. Josephs, R.D., Schuhmacher, R., Krska, R. 2001. International interlaboratory study for the Fusarium mycotoxins zearalenone and deoxynivalenol in agricultural commodities. Food Addit Contam. 18, 417–430. Kallela, K. and Vasenius, L. 1982. The effects of rumen fluid on the content of zearalenone in animal fodder. Nord Vet Med. 34, 336–339. Kang, H.J., Kim, J.C., Sea, J.A., Lee, Y.W., Son, D.H. 1994. Contamination of Fusarium mycotoxins in corn samples imported from China. Agric Chem Biotechnol. 37, 385–391. Kiessling, K.H. and Pettersson, H. 1978. Metabolism of zearalenone in rat liver. Acta Pharmacol Toxicol. 43, 285–290. Kleinova, M., Zöllner, P., Kahlbacher, H., Hochsteiner, W., Lindner, W. 2002. Metabolic profiles of the zearalenone and of the growth promoter zeranol in urine, liver, and muscle of heifers. J Agric Food Chem. 50, 4769–4776. Kollarczik, B., Garels, M., Hanelt, M. 1994. In vitro transformation of the Fusarium mycotoxins deoxynivalenol and zearalenone by the normal gut microflora of pigs. Nat Toxins. 2, 105–110. Krska, R. and Josephs, R. 2001. The state-of-the-art in the analysis of estrogenic mycotoxins in cereals. Fresenius’ J Anal Chem. 369, 469–476.

Krska, R. 1998. Performance of modern sample preparation techniques in the analysis of Fusarium mycotoxins in cereals. J Chromatogr A. 815, 49–57. Krska, R., Welzig, E., Boudra, H. 2007. Analysis of Fusarium toxins in feed. Anim Feed Sci Technol. 137, 241–264. Kruger, S.C., Kohn, B., Ramsey, C.S., Prioli, R. 1999. Rapid immunoaffinity-based method for determination of zearalenone in corn by fluorometry and liquid chromatography. J AOAC Int. 82, 1364–1368. Kuiper-Goodman, T., Scott, P.M., Watanabe, H. 1987. Risk assessment of the mycotoxin zearalenone. Regul Toxicol Pharmacol. 7, 253–306. Labuda, R., Parich, A., Berthiller, F., Tancinova, D. 2005. Incidence of trichothecenes and zearalenone in poultry feed mixtures from Slovakia. Int J Food Microbiol. 105, 19–25. Lagana, A., Bacaloni, A., Castellano, M., Curini, R., De Leva, I., Faberi, A., Materazzi, S. 2003. Sample preparation for determination of macrocyclic lactone mycotoxins in fish tissues, based on on-line matrix solid-phase extraction cleanup followed by liquid chromatography/tandem mass spectrometry. J AOAC Int. 86, 729–736. Lagana, A., Faberi, Fago., G., Marino, A., Pastorini, E., Samperi, R. 2004. Application of an innovative matrix solid phase extraction liquid chromatography tandem mass spectrometry analytiocal methodology to the study of the metabolism of the estrogenic mycotoxin zearalenone in rainbow trout liver and muscular tissue. Int J Environ Anal Chem. 84, 1009–1016. Larriuz-Serrano, M.C., Perez-Cardona, C.M., RamosValencia, G., Bourdony, C.J. 2001. Natural history and incidence of premature thelarche in Puerto Rican girls aged 6 months to 8 years diagnosed between 1990 and 1995. P. R. Health Sci. J. 20, 13–18. Launay, F.M., Young, P.B., Sterk, S.S., Blokland, M.H., Kennedy, D.G. 2004. Confirmatory assay for zeranol, taleranol, and the Fusarium spp. toxins in bovine urine using liquid chromatography-tandem mass spectrometry. Food Addit Contam. 21, 52–62. Lee, M.G., Yan, Q.M., Hart, L.P., Pestka, J.J. 2001. Enzyme-linked immunosorbent assays of zearalenone using polyclonal, monoclonal, and recombinant antibodies. Methods Mol Biol. 157, 159–170. Llorens, A., Mateo, R., Mateo, J.J., Jimenez, M. 2002. Comparison of extraction and clean-up procedures for analysis of zearalenone in corn, rice, and wheat grains by high-performance liquid chromatography with photodiode array and fluorescence detection. Food Addit Contam. 19, 272–281. Llorens, A., Mateo, R., Hinojo, M.J., Logrieco, A., Jimenez, M. 2004. Influence of the interactions among ecological variables in the characterization of zearalenone producing isolates of Fusarium spp. Syst Appl Microbiol. 27, 253–260. Malekinejad, H., Colenbrander, B., Fink-Gremmels, J. 2006. Hydroxysteroid dehydrogenases in bovine and porcine granulosa cells convert zearalenone into its hydroxylated metabolites alpha-zearalenol and betazearalenol. Vet Res Commun. 30, 445–453. Maragos, C.M. and Kim, E.K. 2004. Detection of zearalenone and related metabolites by fluorescence

Mycoestrogens

polarization immunoassay. J Food Prot. 67, 1039– 1043. Medina, M.B. and Sherman, J.T. 1986. High performance liquid chromatography separation of anabolic oestrogens and ultraviolet detection of 17 betaoestradiol, zeranol, diethylstilboestrol or zearalenone in avian muscle tissue extracts, Food Addit Contam. 3, 263–272. Meyer, K., Usleber, E., Dietrich, R., Martlbauer, E., Bauer, J. 2002. Zearalenone metabolites in bovine bile. Archiv für Lebensmittelhygiene. 53, 115–117. Milano, G.D. and Lopez, T.A. 1991. Influence of temperature on zearalenone production by regional strains of Fusarium graminearum and Fusarium oxysporum in culture. Int J Food Microbiol. 13, 329–333. Minervini, F. and Dell’Aquila, M.E. 2008. Zearalenone and reproductive function in farm animals. Int J Mol Sci. 9, 2570–2584. Mirocha, C.J., Robinson, T.S., Pawlosky, R.J., Len, N.K. 1982. Distribution and residue determination of (3H) zearalenone in broilers. Toxicol Appl Pharmacol. 66, 77–87. Mirocha, C.J., Pathre, S.V., Robinson, T.S. 1981. Comparative metabolism of zearalenone and transmission into bovine milk. Food Cosmet Toxicol. 19, 25–30. Molto, G.A., Gonzalez, H.H., Resnik, S.L., Pereyra Gonzalez, A. 1997. Production of trichothecenes and zearalenone by isolates of Fusarium spp. from Argentinian maize. Food Addit Contam. 14, 263– 268. Montani, M.L., Vaamonde, G., Resnik, S.L., Buera, P. 1988. Influence of water activity and temperature on the accumulation of zearalenone in corn. Int J Food Microbiol. 6, 1–8. Nuryono, N., Noviandi, C.T., Bohm, J., Razzazi-Fazeli, E. 2005. A limited survey of zearalenone in Indonesian maize-based food and feed by ELISA and high performance liquid chromatography. Food Control. 16, 65–71. Ofitserova, M., Nerkar, S., Pickering, M., Torma, L., Thiex, N. 2009. Multiresidue mycotoxin analysis in corn grain by column high performance liquid chromatography with postcolumn photochemical and chemical derivatization: Single laboratory validation. J Off Anal Chem. 92, 15–25. Oliveira, G.R., Ribeiro, J.M., Fraga, M.E., Cavaglieri, L.R., Direito, G.M., Keller, K.M., Dalcero, A.M., Rosa, C.A. 2006. Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins, and zearalenone in the Rio de Janeiro state, Brazil. Mycopathologia. 162, 355–362. Olsen, M., Pettersson, H., Sandholm, K., Visconti A., Kiessling, K.-H. 1987. Metabolism of zearalenone by sow intestinal mucosa in vitro. Food Chem Toxicol. 25, 681. Olsen, M., Malmlof, K., Pettersson, H., Sandholm, K., Kiessling, K.H. 1985. Plasma and urinary levels of zearalenone and alpha-zearalenol in a prepubertal gilt fed zearalenone. Acta Pharmacol Toxicol. 56, 239–243. Olsen, M., Pettersson, H., Kiessling, K.H. 1981. Reduction of zearalenone in female rat liver by 3

241

alpha-hydroxysteroid dehydrogenase. Acta Pharmacol Toxicol. 48, 157–161. Ouanes, Z., Ayed-Boussema, I., Baati, T., Creppy, EE., Bacha, H. 2005. Zearalenone induces chromosome aberrations in mouse bone marrow: preventive effect of 17beta estradiol, progesterone and vitamin E. Mut. Res. 565, 139–149. Pallaroni, L. and von Holst, C. 2003. Comparison of alternative and conventional extraction techniques for the determination of zearalenone in corn. Anal Bioanal Chem. 376, 908–912. Pallaroni, L., von Holst, C., Eskilsson, C.S., Bjorklund, E. 2002. Microwave-assisted extraction of zearalenone from wheat and corn. Anal Bioanal Chem. 374, 161–166. Pallaroni, L., Björklund, E., and von Holst, C. 2002. Optimization of atmospheric pressure chemical ionization interface parameters for the simultaneous determination of deoxynivalenol and zearalenone using hplc/ms. J Liq Chromatogr Relat Technol. 25, 913–926. Pichler, H., Krska, R., Szekacs, A., Grasserbauer, M. 1998. An enzyme-immunoassay for the detection of the mycotoxin zearalenone by use of yolk antibodies. Fresenius’ J of Anal Chem. 362, 176–177. Plasencia, J. and Mirocha, C.J. 1991. Isolation and characterisation of zearalenone sulphate produced by Fusarium spp. Appl Environ Microbiol. 57, 146– 150. Pfohl-Leszkowicz, A., Chekir-Ghedira L., Bacha H. 1995. Genotoxicity of zearalenone, an estrogenic mycotoxin: DNA adduct formation in female mouse tissues. Carcinogenesis. 16, 2315–2320. Quiroga, N., Sola, I., Varsavsky, E. 1994. Selection of a simple and sensitive method for detecting zéaralenone in corn. J Assoc Anal Chem Int. 77, 939–941. Rosenberg, E., Krska, R., Wissiack, R., Kmetov, V., Josephs, R., Razzazi, E., Grasserbauer, M. 1998. Highperformance liquid chromatography-atmosphericpressure chemical ionization mass spectrometry as a new tool for the determination of the mycotoxin zearalenone in food and feed. J of Chromatogr. 819, 277–288. Ryu, D. and Bullerman, L.B. 1999. Effect of cycling temperatures on the population of deoxynivalenol and zearalenone by Fusarium graminearum NRRL 5883. J Food Prot. 62, 1451–1455. Ryu, J.C., Yang, J.S., Song, Y.S., Kwon, O.S., Park, J., Chang, I.M. 1996. Survey of natural occurrence of trichothecene mycotoxins and zearalenone in Korean cereals harvested in 1992 using GC-MS. Food Addit Contam. 13, 333–341. Saenz de Rodriguez, C.A. 1984. Environmental hormone contamination in Puerto Rico. N. Engl. J. Med. 310, 1741–1742. Saenz de Rodriguez, C.A., Bongiovanni, A.M., Conde de Borrego, L. 1985. An epidemic of precocious development in Puerto Rican children. J. Pediatr. 107, 393–396. Schollenberger, M., Muller, H.M., Rufle, M., Suchy, S., Planck, S., Drochner, W. 2005. Survey of Fusarium toxins in foodstuffs of plant origin marketed in Germany. Int J Food Microbiol. 97, 317–326.

242

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Schothorst, R.C. and Van Egmond, H.P. 2004. Report from SCOOP task 3.2.10: Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states: Subtask: Trichothecenes. Toxicol Lett. 153, 133–143. Scott, P.M. 1995. Official Methods of Analysis of AOAC International, 16th ed., AOAC International. Arlington, VA, sec 985.18. Scott, P.M. and Lawrence, G.A. 1988. Liquid chromatographic determination of zearalenone and alpha and beta zearalenol in milk. J Assoc Off Chem. 71, 1176– 1179. Scudamore, K.A. and Livesey, C.T. 1998. Occurrence and significance of mycotoxins in forage crops and silage: A review. J Sci Food Agric. 77, 151–157. Shim, W.B., Dzantiev, B.B., Eremin, S.A., Chung, D.H. 2009. One-step simultaneous immunochromatographic strip test for multianalysis of ochratoxin A and zearalenone. J Microbiol Biotechnol. 19, 83–92. Silva, C.M. and Vargas, E.A. 2001. A survey of zearalenone in corn using Romer Mycosep 224 column and high performance liquid chromatography. Food Addit Contam. 18, 39–45. Songsermsakul, P., Sontag, G., Cichna-Markl, M., Zentek, J., Razzazi-Fazeli, E. 2006. Determination of zearalenone and its metabolites in urine, plasma, and feces of horses by HPLC-APCI-MS. J Chromatogr B. 843, 252–261. Stob, M., Baldwin, R.S., Tuite, J., Andrews, F.N., Gillette, K.G. 1962. Isolation of an anabolic, uterotrophic compound from corn infected with Gibberella zeae. Nature. 29, 196. Stopper, H., Schmitt, E., Kobras, K. 2005. Genotoxicity of phytoestrogens. Mutat Res. 574, 139–155. Sundlof, S.F. and Strickland, C. 1986. Zearalenone and zeranol : Potential residue problems in livestock. Vet Hum Toxicol. 28, 242–250. Sydenham, E.W., Marasas, W.F., Thiel, P.G., Shepard, G.S., Nieuwenhuis, J.J. 1991. Production of mycotoxins by selected Fusarium graminearum and F. crookwellense isolates. Food Addit Contam. 8, 31–41. Takemura, H., Shim, J.Y., Sayama, K., Tsubura, A., Zhu, B.T., Shimoi, K. 2007. Characterization of the estrogenic activities of zearalenone and zeranol in vivo and in vitro. J Steroid Biochem Mol Biol. 103, 170–177. Tanaka, T., Yoneda, A., Inoue, S., Sugiera, Y., Ueno, Y. 2000. Simultaneous determination of trichothecene mycotoxins and zearalenone in cereals by gas chromatography-mass spectrometry. J Chromatogr. 882, 23–28. Tanaka, T., Hasegawa, A., Matsuki, Y., Lee, U.S., Ueno, Y. 1985. Rapid and sensitive determination of zearalenone in cereals by high performance liquid chromatography with fluorescence detection. J Chromatogr. 328, 271–278. Tanaka, T., Teshima, R., Ikebuchi, H., Sawada, J., Terao, T., Ichinoe, M. 1993. Sensitive determination of zearalenone and alpha zearalenol in barley and Job’s tears by liquid chromatography with fluorescence detection. J AOAC Int. 76, 1006–1009.

Thongrussamee, T., Kuzmina, N.S., Shim, W.B., Jiratpong, T., Eremin, S.A., Intrasook, J., Chung, D.H. 2008. Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals. Food Addit Contam. 25, 997–1006. Turner, G.V., Phillips, T.D., Heidelbaugh, N.D., Russell, L.H. 1983. High pressure liquid chromatographic determination of zearalenone in chicken tissues. J Assoc Off Anal Chem. 66, 102–104. Visconti, A., and Pascale, M. 1998. Determination of zearalenone in corn by means of immunoaffinity clean up and high performance liquid chromatography with fluorescence detection. J Chrom A. 815, 133– 140. Ware, G.M., Yuhong Zhao, S.S., Kuan, S.S., Carman, A.S. 1999. Preparative method for isolating αzearalenol and zearalenone using extracting disk. J of AOAC Int. 82, 90–94. Weiss, R., Freundenschuss, M., Krska, R., Mizaikoff, B. 2003. Improving methods of analysis for mycotoxins: Molecularly imprinted polymers for deoxynivalenol and zearalenone. Food Addit Contam. 20, 386– 395. Xia, X., Li, X., Ding, S., Zhang, S., Jiang, H., Li, J., Shen, J. 2009. Ultra-high pressure liquid chromatographytandem mass spectrometry for the analysis of six resorcylic acid lactones in bovine milk. J Chrom A. 1216, 2587–2591. Zhang, W., Wang, H., Wang, J., Li, X., Jiang, H., Shen, J. 2006. Multiresidue determination of zeranol and related compounds in bovine muscle by gas chromatography/mass spectrometry with immunoaffinity cleanup. J AOAC Int. 89, 1677–1681. Zinedine, A., Soriano, J.M., Molto, J.C., Manes, J. 2007. Review on the toxicity, occurrence, metabolism, detoxification, regulations, and intake of zearalenone: An oestrogenic mycotoxin. Food Chem Toxicol. 45, 1–18. Zöllner, P., Berner, D., Jodlbauer, J., Lindner, W. 2000. Determination of zearalenone and its metabolites αand β-zearalenol in beer samples by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 738, 233–241. Zöllner, P., Jodlbauer, J., Kleinova, M., Kahlbacher, H., Kuhn, T., Hochsteiner, W., Lindner, W. 2002. Concentration levels of zearalenone and its metabolites in urine, muscle tissue, and liver samples of pigs fed with mycotoxin-contamined oats. J Agric Food Chem. 50, 2494–2501. Zöllner, P., Jodlbauer, J., Lindner, W. 1999. Determination of zearalenone in grains by high-performance liquid chromatography-tandem mass spectrometry after solid-phase extraction with RP-18 columns or immunoaffinity columns. J Chromatogr. 858, 167–174. Zöllner, P. and Mayer-Helm, B. 2006. Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry. J Chromatogr A. 1136, 123–169.

Chapter 9 Analysis of Hormones in Food John L. Zhou and Zulin Zhang

Introduction Endocrine-disrupting chemicals (EDCs) are environmental contaminants that interfere with the function of the endocrine system of wildlife and humans. There are over 38,000 chemicals suspected of causing endocrine disruption, including estrogens, personal pharmaceutical products, flame retardants, pesticides (Shi et al. 2007; Gabet et al. 2007; Wang et al. 2008). There is evidence that domestic animals and wildlife have suffered adverse consequences from exposure to EDCs. Literature reports that there are changes in sex steroids in fish, abnormal reproductive development in alligators, and birth defects in Lake Michigan cormorants (large seabirds), as well as other problems (Kuster et al. 2004; Shi et al. 2007; Gabet et al. 2007; Wang et al. 2008). The range of estrogens reported to cause endocrine disruption is diverse and includes both natural and synthetic chemicals. Naturally produced estrogens such as estrone (E1) and 17β-estradiol (E2) are mainly derived from excreta of humans and livestock. It has been shown that a woman can excrete 7 μg of E1 and 2.4 μg of E2 per day (Adlercreutz et al. 1986; Sumpter and Jobling 1995). Man-made substances include synthetically produced hormones, such as 17α-ethynylestradiol (EE2), which is an Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

ingredient of the human contraceptive pill (Desbrow et al. 1998; Liu et al. 2004). The estrogenic potency of these three sterols is three or more orders of magnitude higher than that of most other EDCs, such as bisphenol A and nonylphenols, which are weakly estrogenic (Shi et al. 2004). These estrogens in the environment can induce tremendous endocrine-disrupting effects, even in a very low concentration. For example, feminization of male fish occurs in the presence of 1–10 ng L−1 E2, or 0.1 ng L−1 of the synthetic estrogen EE2 (Wang et al. 2008). The discharged domestic effluents represent the most significant estrogenic input to the environment and serve as important point sources, especially in densely populated areas (Belfroid et al. 1999). Among the three sterols, EE2 showed the highest estrogenic potency in in vitro tests, followed by E2, then E1 (Larsson et al. 1999). In addition, EE2 is considerably more persistent in wastewater treatment plants compared to the other natural hormones. Due to the introduction of the ethynyl group, the ring of EE2 becomes extremely stable against oxidation. Estrogens, like all steroids, share the same hydrocarbon ring nucleus as cholesterol, their parent compound. Their fate and behavior are therefore influenced by their physicochemical properties, which are summarized in Table 9.1. The log octanol-water partition coefficient (Kow) values are 3.43 for E1, 3.94 for E2, and 4.15 for EE2, and thus it is evident that these compounds are slightly lipophilic and have limited solubility in water. All these 243

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Table 9.1. Selected physicochemical properties of E1, E2, EE2, and E3. Chemical Name E1 E2 EE2 E3

Molecular Weight

Water Solubility (mg L−1)

Vapor Pressure (mm Hg)

log Kow

270.4 272.4 296.4 288.4

13 13 4.8 13.3

2.3 × 10−10 2.3 × 10−10 4.5 × 10−10 6.8 × 10−15

3.43 3.94 4.15 2.8

steroids have very low vapor pressure, ranging from 2.3 × 10−10 to 6.8 × 10−10 mm Hg, indicating low volatility of these compounds (Shi et al. 2007; Wang et al. 2008). The log Kow values of 3.43–4.15 indicate that estrogens should appreciably adsorb into organic matter. It was reported that steroid hormones have been found in fish and poultry, as well as in other animal-derived food such as eggs and milk (Wang et al. 2008). Through intake of contaminated fish, the food chain becomes an important and risky exposure pathway for humans. The contribution of dietary exposure will vary as a function of dietary preferences, position in the food chain, and species and quantities consumed. Studies have shown that lipophilic, persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethanes (DDTs) often bioaccumulate in species at the top level of the food chain, although similar research is limited on hormones. Fish-eating birds and marine mammals have been found to have concentrations of POPs many times higher than those found in fish on which they feed or compared with the levels in the surrounding waters. In some cases, the concentrations of hormones can be elevated by a factor of hundreds or millions (Damstra et al. 2002) through biomagnification in the food web. Human dietary exposure to chemicals via food can be assessed directly through chemical analysis of food items in duplicate diet studies or indirectly through market basket or total diet surveys (e.g., food diaries, food frequency questionnaires). Sensitive and specific methods are therefore needed to detect EDCs from complex matrices. The

choice of method for the determination of EDCs depends on the required outcome. Biological methodologies ascertain the endocrine-disrupting activity exhibited by a chemical or sample, and chemical techniques identify known chemicals and quantify their concentrations within that sample (Gomes et al. 2003). Enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) can be used for multianalyte screening of a series of analytes (Van Oosthuyze et al. 1997; Haasnoot et al. 2002). Instrumental analysis methods used for screening and confirmation, such as gas chromatography (GC) and highperformance liquid chromatography (HPLC) coupled with mass spectrometry (MS) are used most frequently (Cai and Henion 1997; Vulliet et al. 2007; Noppe et al. 2005). In this chapter, we will review the chemical and biological methods that are widely used for the analysis of hormones in food samples, including the sample extraction, cleanup, instrumental analysis, and quality control (QC).

Sample extraction Traditionally, the extraction of estrogens from food samples, such as fish, meat, egg, and animal tissue, has been conducted with organic solvents. Such methods are laborious and time consuming. Unconjugated estrogens have a low aqueous solubility (4.8–13.3 mg L−1) and are easily dissolved in organic solvents. Extraction is generally conducted with ethyl acetate (Markman et al. 2007), methanol or ethanol (Slikker et al. 1982), acetonitrile (Fuh et al. 2004), acetone (Ternes et al. 2002), hexane (Fernandez et al. 2004), methylene chloride, and so on. Some organic solvents

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also denature the sample protein, resulting in a cleaner extraction and helping to release hormone residues bound to proteins. Liquid extraction methods such as Soxhlet extraction (SE), ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), and pressurized liquid extraction (PSE) or accelerated solvent extraction (ASE), as often named, have been used for extraction of typical hormones from solid matrices such as biological and tissue samples. SE is a classical extraction method with proven efficiency, but it takes a long time (usually 10–24 h) and uses a large volume of solvents (normally 200–300 mL). Automatic SE is a modification of the traditional approach, requires less extraction time and solvents, and can extract four or more samples simultaneously. After extraction, a selective cleanup process is indispensable to the removal of the coextracts due to the high lipid content of biological samples. Gel permeation chromatography (GPC) and multistep column chromatography (using absorbents such as silica gel, alumina, polyamide, and Florisil) have been reported (Meier et al. 2005). The GPC system requires a large volume of solvents, which are expensive and time consuming (Wang et al. 2007), and the target chemicals are easily lost during the tedious elution processes of multistep column chromatography (Ternes et al. 2002). Commercial extraction cartridges containing suitable adsorbents are more convenient for operation and good selective enrichment performance. Liu et al. (2009) used SE followed by cleanup with a silica gel cartridge for GC-MS determination of the target EDCs in biological tissue samples, and the method was successfully applied to real sample analysis. For the optimized method, the recovery efficiencies of E1, E2, EE2, and E3 were from 79.7% to 84.5%, with the relative standard derivations (RSDs) less than 12.1%. Liu et al. (2004) applied MAE followed by silica gel cleanup and analysis by GC-MS for the simultaneous extraction and determina-

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tion of contrasting EDCs, including E1, E2, and EE2, in solid matrices. The results showed that the most efficient extraction (recovery >74%) of the target compounds was achieved by using methanol as the solvent, extraction temperature of 110°C, and 15 min of holding time. The limits of detection (LOD) varied between 0.2 and 1.0 ng g−1 dry weight, and the method was successfully used to extract and analyze EDCs from environmental samples. Labadie and Hill (2007) also used MAE as an extraction technique followed by solid-phase extraction (SPE) cleanup and analysis by LC-MS/MS for E1, E2, and EE2 determination. The whole-procedure recoveries ranged from 82% to 98% for these three estrogens. The method LOD achieved (MAE-SPE and LC-MS/MS) was 15, 30, and 40 pg g−1 for E1, E2, and EE2, respectively, which was 13-fold lower than that obtained by LC-time of flight (TOF)-MS. ASE is a modern extraction method introduced in 1995 that has significantly streamlined sample preparation for solid and semisolid food samples (Eljarrat and Barcelo 2004). It allows a reduction of both extraction time and organic solvent consumption and increases sample throughout. Typically, it will take only 15 min per sample, which is much faster than Soxhlet (6 h per sample; Weltin et al. 2004). ASE automatically separates extracts so that no centrifugation or decantation needs to be applied as is often required for sonication or batched extracts. Its application has been reported in several studies with different objectives and matrices (Ramos et al. 2002). ASE has been used successfully for the measurement of a wide range of organic compounds in a variety of samples, including PCBs and organochlorine pesticides in animal tissues (Kania-Korwel et al. 2008; Suchan et al. 2004) and polybrominated diphenyl ethers (PBDEs) have been used in biological samples (Tapie et al. 2008). In addition, ASE has been shown to represent a rapid, effective, and reliable technique for extracting hormones from solid samples (Chun et al.

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2005; Beck et al. 2008), which could be followed by SPE cleanup and GC-MS analysis. Beck et al. (2008) compared different solvents and optimized the PLE parameters when investigating estrogen extraction. The results (Beck et al. 2008) showed that better recoveries were obtained by using acetone (89– 103%) and ethyl acetate (71–100%) than by other solvents such as dichloromethane (5– 46%), methanol (1–8%), and hexane/acetone (1:1, v/v; 15–21%). The other solvents and solvent mixtures need more than one extraction cycle for complete estrogen extraction. Overall, mixed solvents did not lead to a higher recovery of estrogen with its OH- and O-functional groups. Minor changes in solvent polarity seem to have a major effect on the extraction efficiency at higher temperatures. Extraction efficiency using acetone reached an optimum at 60°C. At higher temperatures, the recoveries were found to decrease, which is probably attributable to a destabilization or destruction of the compounds or increased formation of nonextractable complexes. Below 60°C, low estrogen recoveries were most probably due to the inefficient desorption and dissolution of the estrogens within 15 min of one extraction cycle. The LOD of the proposed GC-MS methods varied from 1 ng mL−1 to 5 ng mL−1 for estrogens in the final extracts (Beck et al. 2008).

Cleanup procedures Because of the complexity of the food sample matrices, a cleanup step is required prior to the chromatographic separation. After extraction, many of the reported analytical methods proceed with a cleanup procedure by SPE or SPE combined with silica gel cartridges. For this purpose, octadecyl (C18) silica, polymeric materials (Oasis), or Strata-X-AW have been employed as stationary phases (Labadie and Hill 2007). In addition, GPC was used for the extract cleanup (Markman et al. 2007). The SPE cartridge with silica gel as packing material is widely used for cleanup

of estrogen extracts. Silica gel is frequently used in preparative chromatography due to its low operating cost and polar selectivity. Organic chemicals with polar functionalities could be retained when introduced to silica gel in a nonpolar mobile phase. After adsorption to silica gel, the target chemicals could be eluted by solvents with a similar polarity whereas other impurities (especially more polar chemicals) are retained on the silica surface. Liu et al. (2009) used Superclean LC-Si SPE cartridges (1 g, 6 mL) for EDC cleanup. The target EDCs were dissolved in a nonpolar solvent (hexane) and loaded to LC-Si cartridges at a flow rate of 2 mL min−1. Then 30% dichloromethane in hexane (20 mL) and 70% dichloromethane in hexane (20 mL) were used as elution solvents. The two elutes were combined for derivatization and GC-MS analysis. The recoveries for this method were from 79.7% to 93.7% for EDCs, and the method’s detection limits ranged from 0.27 to 0.68 ng g−1 dry weight, which was successfully applied to analysis of the mollusk tissues from Dapeng Bay of China with the concentrations from 1.6 to 131.5 ng g−1. Labadie and Hill (2007) used Strata-X-AW cartridges (Phenomenex, Macclesfield, UK) for sample cleanup, previously conditioned with 5 mL of ethyl acetate, methanol, and water in sequence. After sample loading, the cartridges were washed with 4 mL of methanol/ pH 7.0 acetate buffer (4:6, v/v) and dried under vacuum for 30 min, and the estrogens were eluted with 7 mL of ethyl acetate. Eluates were evaporated under vacuum and reconstituted in 500 μL of cyclohexane/ethyl acetate (9:1, v/v). Further cleanup of these extracts was achieved using silica gel cartridges (500 mg, Sigma-Aldrich, Dorset, UK). Anhydrous sodium sulfate (0.5 g) was packed on top of the silica cartridge, and the sorbent was preconditioned in sequence with 4 mL of cyclohexane/ethyl acetate (6:4, v/v) and 4 mL of cyclohexane. After sample loading, cartridges were washed with 4 mL of cyclohexane and estrogens were eluted with 6 mL of

Analysis of Hormones in Food

cyclohexane/ethyl acetate (6:4, v/v). The extracts were dried, reconstituted in 60 μL of water/acetonitrile (7:3, v/v), and passed through 0.22-μm centrifuge filters. The filters were further rinsed with 20 μL of water/ acetonitrile (7:3, v/v), with the combined extracts ready for further analysis by LC-MS or derivatization followed by GC-MS. Furthermore, hormone extracts can be purified by GPC to remove the large pool of high–molecular-weight lipids, which are unavoidably extracted together with target compounds. Different GPC columns can be used: PTFE and elution with acetone/cyclohexane (1:3, v/v) (Ternes et al. 2002); SX-3 and elution with dichloromethane/hexane (1:1, v/v) (Gomes et al. 2004); and Sephadex LH-20 and elution with dichloromethane/methanol (1:1, v/v) (Isobe et al. 2006). Markman et al. (2007) used a GPC solvent-resistant column (400 mm × 25 mm) from Omnifit (Cambridge, UK), which was packed with 70 g of BioBeads S-X3 preswollen in and washed with cyclohexane/acetone (3:1, v/v). The column was operated under gravity flow conditions at a flow rate of 4 mL min−1, with cyclohexane/ acetone (3:1, v/v) as eluent. The resolution of the system was evaluated using 200 μL of fish oil spiked with a mixture of estrogen standard solutions. The first 97-mL fraction (0–24 min separation) was found to contain only lipid and was discarded in further preparations. The analytes were recovered in the following 48-mL fraction (25–37 min separation) and subjected to further analysis. No compounds of interest were detected in subsequent fractions, and the GPC system was ready to use for the next sample after subsequent elutions with 50 mL of eluent, 50 mL of toluene/ chloroform/methanol (2:2:1, v/v/v), and a further 50 mL of eluent. Reagent blank and blank runs (i.e., the blank extraction was performed with all procedures identical except for the omission of sample material) between each sample chromatography were analyzed to assess potential contamination and carry-

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over in the system. The blank value was subtracted from the corresponding sample, and the significance of differences between blank and sample values was discussed using the three-standard deviation rule for blank correction (e.g., corresponding to the average signal from blank samples plus three standard deviations). Further purification, when performed, could be carried out by HPLC (Ternes et al. 2002; Peck et al. 2004; Williams et al. 2003) or using restricted-access materials (RAMs) (Petrovic et al. 2002). RAMs are bifunctional sorbents that combine size exclusion and reversed-phase retention mechanisms, tailored for the separation of macromolecular matrix components and the adsorption of low-molecular target analytes, all in one step. Preparative HPLC fractioning, using reversed-phase C18 columns, has been included in some preparation procedures as a purification step preceding GC-MS analysis (Ternes et al. 2002; Peck et al. 2004; Williams et al. 2003). It is difficult to compare the performance of the different techniques. Nevertheless, we note that the most widespread in use, and the simplest system, is SPE, which gives satisfactory recoveries for different matrices. SPE purification with silica gel, which represents the most common purification technique for the analysis of hormones from various matrices by LC-MS/MS, leads to good performance of 90–100% recoveries (Gabet et al. 2007). It is applicable to all kinds of matrices. GPC is used for solid matrices, with analysis either by LC-MS/MS or GC-MS/MS, and is generally coupled with SPE (Gabet et al. 2007).

Instrumental analysis In terms of food analysis applications, LC-MS/MS, GC-MS, and GC-MS/MS are the most widely used for the analysis of hormones among different analytical techniques.

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Liquid chromatography LC is performed mainly in reversed phase, with the MS as the most widely used detector. In the MS, electrospray interface (ESI) in negative ion (NI) mode is generally used as the ionization method. Alternatively, some studies reported the use of ESI and atmospheric pressure chemical ionization (APCI) in positive mode. Silica-based reversed-phase columns, such as C18, have been chosen more often for separation. The mobile phases mainly consist of acetonitrile and water mixtures, methanol and water mixtures, or ternary mixtures of acetonitrile, methanol, and water. Sometimes, the mobile phases are acidified with 0.5% acetic acid (Brossa et al. 2004; Sole et al. 2000) to render acidic compounds into nonionized form. Several spectroscopic techniques, such as ultraviolet (UV), fluorescence, photodiode array (PDA), and diodearray (DAD), as well as MS, have been used in LC analysis of hormones (Wang et al. 2008). In the past, UV was almost exclusively used. More recently, LC-MS techniques have become more accessible, hence more popular and widely used in food analysis. LC-MS, unlike GC-MS, is not limited by such factors as nonvolatility and high molecular weight, and it enables the determination of both conjugated and nonconjugated estrogens without the need for derivatization or hydrolysis. Also, one study reported the use of DAD (Almeida and Nogueira 2006) for hormone analysis. The LODs obtained using DAD are higher (parts per billion range [ppb]) than those with MS/MS detection (LOD under or in the range of parts per trillion [ppt], depending on matrices and analytes). In general, LC is less widely used than GC for the analysis of solid matrices (Gabet et al. 2007). LC-MS has often been carried out with ESI operated in the negative ion (NI) mode or APCI. Another benefit of LC-MS method is the possibility of integrating sample preparation and enrichment online with the analy-

sis. In addition, the emergence of LC-MS/MS has significantly improved the performance of the technique by improving the detection and quantification limits and enhancing analyte identification. In order to achieve sufficient sensitivity, most of these methods are based on LC-MS and LC-MS/MS. However, they are not accessible in all laboratories for routine analysis due to the high cost of equipment and the requirement for an experienced operator. Thus, LC is a good choice for which the sample pretreatment step prior to chromatographic analysis is usually unnecessary, except to improve the limits of sensitivity and selectivity (Wang et al. 2008).

Gas chromatography Many studies have shown that GC-MS or GC-MS/MS have been widely applied for monitoring estrogens with low LOD of ppt or sub-ppt level. In order to decrease the polarity of the target estrogens and enhance the sensitivity and effect of the column, silylation and acetylation are often used before quantitative determination by GC or GC-MS. Different agents have been used for silylation, and the N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) is used most frequently. Other agents are also used, such as N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA), N-trimethylsilyl acetamide (TMSA), and N-(tert-butyldimethylsilyl) - N - methyl - trifluoroacetamide (MTBSTFA). They can be used alone or in combination with a small proportion of different catalysts, such as N-methyl-N(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (MSTFA/TMCS), MSTFA/ trimethylsilylimidazole (TMIS), BSTFA/ TMCS/TMSI, MSTFA/TMSI/dithioerytol (DTE), and pentafluorobenzoyl chloride (PFBCI), which have been employed for the derivatization of the hydroxyl groups contained in the estrogen moiety. Acetylation with anhydrides, such as heptafluorobutyric anhydride and pentafluoropropionic acid

Analysis of Hormones in Food

anhydride (PFPA), is the other frequently applied derivatization technique. More importantly, the humic and fulvic acids in the water samples may disturb the derivatization. Both increasing the quantity of agents for derivatization and prolonging the time of reaction can enhance the rate of products. LODs for GC-MS-based methods are typically above ppt, resulting in a significant number of “no detections” in the data reported using these methods. Methods based on GC-MS/MS are more sensitive than GC-MS but still require the derivatization step prior to analysis. GC with electron capture detector (ECD) has also been used. Pinnella et al. (2001) isolated the catecholestrogens from E2 by SPE, after derivatizing and subjecting them to solvent exchange prior to analysis. The LODs of the method are 0.8 and 1.3 ng mg−1, and the limits of quantification (LOQs) are 2.6 and 4.3 ng mg−1. The use of GC solely for the determination of estrogens is rare, and GC-MS and GC-MS/MS, as well as LC-MS and LC-MS/MS, are frequently used. Most of the development in the GC-based technique is concerned with the improvement in silylation and acetylation.

Other techniques Enzyme-linked immunosorbent assay and radioimmunoassay An enzyme-linked immunosorbent assay (ELISA) for detection of estrogens has been developed and is especially suitable for screening large numbers of hormonally active substances with a great sensitivity. Unlike an radioimmunoassay (RIA), in which a radioactive isotope is used, ELISA does not entail serious problems in the disposal of samples, and it does not require the use of reagents that are harmful to human health (Wang et al. 2008). Monteverdi and Di Giulio (1999) developed a method using primary hepatocyte cultures from the channel catfish (Ictalurus

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punctatus) with an ELISA to detect and quantify the production of vitellogenin (VTG), a liver-derived estrogen-induced lipoprotein; the LOD for this assay was typically 15–25 ng VTG mL−1 medium. Nash et al. (2000) developed a simple and rapid ELISA, which used acetylcholinesterase tracer to increase the sensitivity of assay (1.6 pg E2 well−1) so that reliable measurements of each steroid could be achieved with only 10 μL of plasma. Typical standard curves show a workable range of 0.8–400 pg well−1. A polyclonal antibody against the highest-molecular-weight band of putative VTG was generated in sheep, and an indirect antibody-capture competitive ELISA was developed. VTG was purified from the plasma of E2-injected male greenback flounder, Rhombosolea tapirina. The LOD of E2 in plasma was 0.3 ng mL−1, and a working range for the standard curve of 0.16– 20 μg mL−1 was chosen on the basis of the linear portion of the displacement curve (Sun et al. 2003). A rare earth ion chelate has a large stokes shift, narrow emission bands, and long fluorescence decay time (over 10,000 times longer than general fluorescence). Therefore, time-resolved fluoroimmunoassay (TR-FIA), which uses a rare earth ion chelate for labeling and detection, is a highly sensitive measurement (Ito et al. 1999). A direct TR-FIA system for measuring E2 in bovine plasma was developed by Takahashi et al. (2004): the sample was detected without prior extraction and purification and the minimum detectable concentration was 0.625 pg well−1. A new fluorometric enzyme immunoassay for E2 using biotinylated estradiol (BE) as a probe ligand was described by Matsumoto et al. (2006). In this method, E2 was detected indirectly by a solid-phase avidin-biotin-binding assay, in which the biotin was immobilized on a microtiter plate. The LOD and linear range for the determination of E2 were 0.12 nmol L−1 and from 0.12 to 25 nmol L−1, respectively. Sometimes RIA can be used to measure very low levels of estrogens in samples

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(Geisler et al. 2000); however, the treatment of samples is slow and complex. RIA is suitable for large numbers of samples, but few published techniques provide reliable measurements when the sample volume is small. Furthermore, just like other immunoassays, RIA also shows cross-reactivity with several natural estrogens (Akpoviroro and Fotherby 1980). Consequently, liquid-liquid extraction (LLE) (Stanczyk et al. 1980; Snyder et al. 1999) and SPE (Tacey et al. 1994), followed by HPLC or an immunoaffinity column (IAC) (Zacur et al. 1991), had to be performed before RIA analysis. Surface plasmon resonance-based (SPR-based) immunoassay is not as sensitive as traditional RIA and ELISA, but this method requires no separation and washing after addition of the antibody, steps that are relatively time consuming (Usami et al. 2002; Miyashita et al. 2005). Geisler et al. (2000) described a new method for simultaneous measurement of the three main estrogen fractions, E1, E2, and estrone sulfate (E1S) in breast tumor tissue. HPLC was used to purify the individual estrogen fractions prior to RIA analysis. The LOD of this method was 4.3 fmol g−1 tissue for E2, 19.8 fmol g−1 tissue for E1, and 11.9 fmol g−1 for E1S. In vitro screening for estrogenic activity has often been performed using traditional receptor-binding assays with radiolabeled E2 (Blair et al. 2000). Because the use of radioisotopes has drawbacks, for example radiation hazards and the need for special facilities, several non-radioactive estrogen receptor-binding assays have been recently developed (Seifert et al. 1999; Ohno et al. 2002; Kuramitz et al. 2002). The homogeneous enzyme immunoassay (HEIA) is simpler and faster, and separation or washing steps are not required. Therefore, HEIA, based on measuring changes in enzyme activity caused by the formation of an immune complex in a solution, is regarded as an excellent strategy for overcoming the disadvantages associated with ELISA (Broyles and

Rechnitz 1986; Zherdev et al. 1997; Karapitta et al. 2001).

Analytical quality control To ensure data quality, all the analytical processes should be subject to strict QC procedures to determine systematic and random errors. QC measures in relation to food sample analysis include the collection of blank samples derived from laboratory-grade noncontaminated food samples to determine if sampling procedures, sampling equipment, field conditions, sample shipment and storage (field blank), or laboratory procedures (laboratory blank) may introduce the target analytes into food samples. The spiked food samples are used to check the method precision and recoveries. The blank and spiked food samples are typically made in similar matrices and are free from contamination. Typically, several blank and spiked samples are produced with each set of real samples (e.g., 10 samples for each set). In addition, the random errors involved in sampling are assessed by carrying out replicate sampling of food samples at the same time and analyzing sample extracts. Internal standards (usually the target compounds labeled by stable isotopes such as 13C or 2H) are used to compensate for losses involved in the sample extraction and workup to further characterize the method’s performance. Prior to use, all glassware is rinsed with dichloromethane (two times) and methanol (two times) or is baked at 450°C for 4 h. All the solvents used are of distilled-in-glass grade. All these processes are carried out to minimize crosscontamination and the loss of analytes through adsorption onto the surface of sampling vessels and the extraction apparatus. Because food samples are diverse in nature and origin, appropriate blanks should be used to target the samples under investigation. To assess systematic errors, most studies would use the so-called recovery experiments by spiking known amounts of each target

Analysis of Hormones in Food

compound in food samples, followed by extraction and analysis. This gives a good indication of how reliable the measurement values are. In addition, certified reference material (CRM) for estrogens in food samples should be prepared that can identify the closeness between a measured value (from an individual laboratory) and a certified value (from the supplier). Because CRM is vigorously tested under varying environmental conditions by the supplier and independently verified by laboratories worldwide, it becomes a calibration tool for the international community for hormone research. Ideally, the relative difference between measured and certified values should be as small as possible (e.g., ±10%). Such material will ensure that everyone follows the right procedures and generates data of the highest quality. In addition, this practice will ensure monitoring data obtained from any food samples tested anywhere can be compared to identify hot spots of hormone pollution in food samples and temporal variation of hormone concentrations in the food samples from different places or regions.

Conclusion This chapter describes the analytical methods for the monitoring of hormones in food samples. The current hormone analysis techniques are primarily based on MS detection or bioassay methods. Methods such as MS are specific and sensitive analytical methods, but they require sample pretreatment procedures. Determination by GC-MS requires that the steroids be derivatized to more volatile forms prior to analysis, whereas LC-MS does not need sample derivatization and hence is used more frequently today. No matter which analytical technique is used, prior sample treatment is often needed, through offline or on-line SPE in most cases. ELISA is a specific and sensitive detection method, and it is simple and economical to use. However, immunoassays also have their

251

disadvantages, such as a significant development time, and poor performance for multiresidue analysis. In addition, they may produce erroneous results due to crossreactions, poor accuracy or reproducibility, and overestimation of trace amounts of hormones caused by nonspecific matrix effects. Thus, a further confirmation analysis using analytical instruments such as GC-MS or LC-MS is often needed to ascertain the results of ELISA.

References Adlercreutz H., Fotsis T., Bannwart C., Hamalainen E., Bloigu S., Ollus A. (1986) Urinary estrogen profile determination in young Finnish vegetarian and omnivorous woman. Journal of Steroid Biochemistry. 24 (1), 289–296. Akpoviroro J. and Fotherby K. (1980) Assay of ethynyloestradiol in human-serum and its binding to plasmaproteins. Journal of Steroid Biochemistry. 13 (7), 773–779. Almeida C. and Nogueira J.M.F. (2006) Determination of steroid sex hormones in water and urine matrices by stir bar sorptive extraction and liquid chromatography with diode array detection. Journal of Pharmaceutical and Biomedical Analysis. 41 (4), 1303–1311. Beck J., Totsche K.U., Kogel-Knabner I. (2008) A rapid and efficient determination of natural estrogens in soils by pressurised liquid extraction and gas chromatography-mass spectrometry. Chemosphere. 71 (5), 954–960. Belfroid A., Van der Horst A., Vethaak A.D., Schafer A.J., Rijs G.B.J., Wegener J., Cofino W.P. (1999) Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in the Netherlands. Science of the Total Environment. 225 (1–2), 101–108. Blair R.M., Fang H., Branham W.S., Hass B.S., Dial S.L., Moland C.L., Tong W.D., Shi L.M., Perkins R., Sheehan D.M. (2000) The estrogen receptor relative binding affinities of 188 natural and xenochemicals: Structural diversity of ligands. Toxicological Sciences. 54 (1), 138–153. Brossa L., Pocurull E., Borrull F., Marce R.M. (2004) Solid phase extraction/high performance liquid chromatography—electrospray mass spectrometry to determine endocrine disruptors in water samples. Chromatographia. 59 (7–8), 419–423. Broyles C.A. and Rechnitz G.A. (1986) Drug antibody measurement by homogeneous enzyme immunoassay with amperometric detection. Analytical Chemistry. 58 (6), 1241–1245. Cai J. and Henion J. (1997) Quantitative multi-residue determination of beta-agonists in bovine urine using on-line immunoaffinity extraction coupled column

252

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packed capillary liquid chromatography tandem mass spectrometry. Journal of Chromatography B. 691 (2), 357–370. Chun S., Lee J., Geyer R., White D.C. (2005) Comparison of three extraction methods for 17β-estradiol in sand, bentonite, and organic rich silt loam. Journal of Environmental Science and Health B. 40, 731–740. Damstra T., Barlow S., Bergman A., Kavlock R., Kraak G.V.D. (2002) Global assessment of the state-ofthe-science of endocrine disruptors. International Programme on Chemical Safety. WHO/PCS/ EDC/02.2. World Health Organization. Desbrow C., Routledge E.J., Brighty G.C., Sumpter J.P., Waldock M. (1998) Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environmental Science and Technology. 32 (11), 1549–1558. Eljarrat E. and Barcelo D. (2004) Sample handling and analysis of brominated flame retardants in soil and sludge samples. Trends in Analytical Chemistry. 23 (10–11), 727–736. Fernandez M.F., Rivas A., Olea-Serrano F., Cerrillo I., Molina-Molina J.M., Araque P., Martinez-Vida J.L., Olea N. (2004) Assessment of total effective xenoestrogen burden in adipose tissue and identification of chemicals responsible for the combined estrogenic effect. Analytical and Bioanalytical Chemistry. 379 (1), 163–170. Fuh M.R., Huang S.Y., Lin T.Y. (2004) Determination of residual anabolic steroid in meat by gas chromatrography-ion trap-mass spectrometer. Talanta. 64 (2), 408–414. Gabet V., Miege C., Bados P., Coquery M. (2007) Analysis of estrogens in environmental matrices. Trends in Analytical Chemistry. 26 (11), 1113–1131. Geisler J., Berntsen H., Lonning P.E. (2000) A novel HPLC-RIA method for the simultaneous detection of estrone, estradiol, and estrone sulphate levels in breast cancer tissue. Journal of Steroid Biochemistry and Molecular Biology. 72 (5), 259–264. Gomes R.L., Scrimshaw M.D., Lester J.N. (2003) Determination of endocrine disrupters in sewage treatment and receiving waters. Trends in Analytical Chemistry. 22 (10), 697–707. Gomes R.L., Avcioglu E., Scrimshaw M.D., Lester J.N. (2004) Steroid estrogen determination in sediment and sewage sludge: A critique of sample preparation and chromatographic/mass spectrometry considerations, incorporating a case study in method development. Trends in Analytical Chemistry. 23 (10–11), 737–744. Haasnoot W., Kemmers-Voncken A., Samson D. (2002) Immunofiltration as sample cleanup for the immunochemical detection of beta-agonists in urine. Analyst. 127 (1), 87–92. Isobe T., Serizawa S., Horiguchi T., Shibata Y., Managaki S., Takada H., Morita M., Shiraishi H. (2006) Horizontal distribution of steroid estrogens in surface sediments in Tokyo Bay. Environmental Pollution. 144 (2), 632–638. Ito K., Oda M., Tsuji A., Maeda M. (1999) Simultaneous determination of alpha-fetoprotein, human chorionic

gonadotropin and estriol in serum of pregnant women by time resolved fluoroimmunoassay. Journal of Pharmaceutical and Biomedical Analysis. 20 (1–2), 169–178. Kania-Korwel I., Zhao H.X., Norstrom K., Li X.S., Hornbuckle K.C., LehmLer H.J. (2008) Simultaneous extraction and clean-up of polychlorinated biphenyls and their metabolites from small tissue samples using pressurized liquid extraction. Journal of Chromatography A. 1214 (1–2), 37–46. Karapitta C.D., Xenakis A., Papadimitriou A., Sotiroudis T.G. (2001) A new homogeneous enzyme immunoassay for thyroxine using glycogen phosphorylase b-thyroxine conjugates. Clinica Chimica Acta. 308 (1–2), 99–106. Kuramitz H., Natsui J., Sugawara K., Itoh S., Tanaka S. (2002) Electrochemical evaluation of the interaction between endocrine disrupter chemicals and estrogen receptor using 17 beta-estradiol labelled with daunomycin. Analytical Chemistry. 74 (3), 533–538. Kuster M., Lopez M.J., de Alda M.J.L., Barcelo D. (2004) Analysis and distribution of estrogens and progestogens in sewage sludge, soils and sediments. Trends in Analytical Chemistry. 23 (10–11), 790–798. Labadie P. and Hill E.M. (2007) Analysis of estrogens in river sediments by liquid chromatography-electrospray ionisation mass spectrometry comparison of tandem mass spectrometry and time-of-flight mass spectrometry. Journal of Chromatography A. 1141 (2), 174– 181. Larsson D.G.J., Adolfsson-Erici M., Parkkonen J., Pettersson M., Berg A.H., Olsson P.E., Forlin L. (1999) Ethinyloestradiol–an undesired fish contraceptive. Aquatic Toxicology. 45 (2–3), 91–97. Liu R., Zhou J.L., Wilding A. (2004) Microwave-assisted extraction followed by gas chromatography-mass spectrometry for the determination of endocrine disrupting chemicals in river sediments. Journal of Chromatography A. 1038 (1–2), 19–26. Liu Y., Guan Y.T., Mizuno T., Tsuno H., Zhu W.P. (2009) A pretreatment method for GC-MS determination of endocrine disrupting chemicals in mollusk tissues. Chromatographia. 69 (1–2), 65–71. Markman S., Guschina I.A., Barnsley S., Buchanan K.L., Pascoe D., Muller C.T. (2007) Endocrine disrupting chemicals accumulate in earthworms exposed to sewage effluent. Chemosphere. 70, 119–125. Matsumoto Y., Kuramitz H., Itoh S., Tanaka S. (2006) New fluorometric enzyme for 17 beta-estradiol by homogeneous reaction using biotinylated estradiol. Talanta. 69 (3), 663–668. Meier S., Klungsoyr J., Boitsov S., Eide T., Svardal A. (2005) Gas chromatography mass spectrometry analysis of alkylphenols in cod (Gadus morhua) tissues as pentafluorobenzoate derivatives. Journal of Chromatography A. 1062 (2), 255–268. Miyashita M., Shimada T., Miyagawa H., Akamatsu M. (2005) Surface plasmon resonance-based immunoassay for 17-beta-estradiol and its application to the measurement of estrogen receptor-binding activity. Analytical and Bioanalytical Chemistry. 381 (3), 667–673.

Analysis of Hormones in Food

Monteverdi G.H., Giulio R.T.D. (1999) An enzymelinked immunosorbent assay for estrogenicity using primary hepatocyte cultrues from the channel catfish (Ictalurus punctatus). Archives of Environmental Contamination and Toxicology. 37 (1), 62–69. Nash J.P., Davail-Cuisset B., Bhattacharvva S., Suter H.C., Le Menn F., Kime D.E. (2000) An enzyme linked immunosorbant assay (ELISA) for testosterone, estradiol, and 17, 20 beta-dihydroxy-4-pregenen3-one using acetylcholinesterase as tracer: Application to measurement of diel patterns in rainbow trout (Oncorhynchus mykiss). Fish Physiology and Biochemistry. 22 (4), 355–363. Noppe H., De Wasch K., Poelmans S., Van Hoof N., Verslycke T., Janssen C.R., De Brabander H.F. (2005) Development and validation of an analytical method for detection of estrogens in water. Analytical and Bioanalytical Chemistry. 382 (1), 91–98. Ohno K., Fukushima T., Santa T., Waizumi N., Tokuyama H., Maeda M., Imai K. (2002) Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization. Analytical Chemistry. 74 (17), 4391–4396. Peck M., Gibson R.W., Kortenkamp A., Hill E.M. (2004) Sediments are major sinks of steroidal estrogens in two United Kingdom rivers. Environmental Toxicology and Chemistry. 23 (4), 945–952. Petrovic M., Tavazzi S., Barcelo D. (2002) Columnswitching system with restricted access pre-column packing for an integrated sample cleanup and liquid chromatographic mass spectrometric analysis of alkylphenolic compounds and steroid sex hormones in sediment. Journal of Chromatography A. 971 (1–2), 37–45. Pinnella K.D., Cranmer B.K., Tessari J.D., Cosma G.N., Veeramachaneni D.N.R. (2001) Gas chromatographic determination of catechloestrogens following isolation by solid-phase extraction. Journal of Chromatography B. 758 (2), 145–152. Ramos L., Kristenson E.M., Brinkman U.A.Th. (2002) Current use of pressurised liquid extraction and subcritical water extraction in environmental analysis. Journal of Chromatography A. 975 (1), 3–29. Seifert M., Haindl S., Hock B. (1999) Development of an enzyme linked receptor assay (ELRA) for estrogens and xenoestrogens. Analytica Chimica Acta. 386 (3), 191–199. Shi J., Fujisawa S., Nakai S., Hosomi M. (2004) Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia-oxidizing bacterium Nitrosomonas europaea. Water Research. 38 (9), 2323–2330. Shi W.X., Delft I.U., Kujawa K. (2007) Estrogens in aquatic environment: A review. In 018530-SWITCH: Sustainable Water Management in the City of the Future of Sixth Framework Programme (2002–2006). Slikker W., Bailey J.R., Newport G.D., Lipe G.W., Hill D.E. (1982) Placental-transfer and metabolism of 17 alpha-ethynylestradiol-17-beta and estradiol17-beta in the rhesus monkey. Journal of Pharmacology and Experimental Therapeutics. 223 (2), 483–489.

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Snyder S.A., Keith T.L., Verbrugge D.A., Snyder E.M., Gross. T.S., Kannan K., Giesy J.P. (1999) Analytical methods for detection of selected estrogenic compounds in aqueous mixtures. Environmental Science and Technology. 33 (16), 2814–2820. Sole M., de Alda M.J.L., Castillo M., Porte C., LadegaardPedersen K., Barcelo D. (2000) Estrogenicity determination in sewage treatment plants and surface waters from the Catalonian area (NE Spain). Environmental Science and Technology. 34 (24), 5076–5083. Stanczyk F.Z., Gale J.A., Goebelsmann U., Nerenberg C., Martin S. (1980) Radioimmunoassay of plasma ethinylestradiol in the presence of circulating norethindrone. Contraception. 22 (5), 457–470. Suchan P., Pulkrabova J., Hajslova J., Kocourek V. (2004) Pressurized liquid extraction in determination of polychlorinated biphenyls and organochlorine pesticides in fish samples. Analytica Chimica Acta. 520 (1–2), 193–200. Sumpter J.P. and Jobling S. (1995) Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environmental Health Perspectives. 103 (2), 173–178. Sun B., Pankhurst N.W., Watts M. (2003) Development of an enzyme-linked immunosorbent assay (ELISA) for vitellogenin measurement in greenback flounder Rhombosolea tapirina. Fish Physiology and Biochemistry. 29 (1), 13–21. Tacey R.L., Harman W.J., Kelly L.L. (1994) Development of a highly sensitive and specific assay for plasma ethinylestradiol using combined extraction, liquid chromatography and radioimmunoassay. Journal of Pharmaceutical and Biomedical Analysis. 12 (10), 1303–1310. Takahashi T., Hamanaka S., Imai K., Hashizume K. (2004) A direct time-resolved fluoroimmunoassay (TR-FIA) for measuring plasma estradiol-17 beta concentrations in cattle. Journal of Veterinary Medical Science. 66 (3), 225–229. Tapie N., Budzinski H., Le-Menach K. (2008) Fast and efficient extraction methods for the analysis of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices. Analytical and Bioanalytical Chemistry. 391 (6), 2169–2177. Ternes T.A., Andersen H., Gilberg D., Bonerz M. (2002) Determination of estrogens in sludge and sediments by liquid extraction and GC/MS/MS. Analytical Chemistry. 74 (14), 3498–3504. Usami M., Mitsunaga K., Ohno Y. (2002) Estrogen receptor binding assay of chemical with a surface plasmon resonance biosensor. Journal of Steroid Biochemistry and Molecular Biology. 81 (1), 47–55. Van Oosthuyze K.E.I., Arts C.J.M., Van Peteghem C.H. (1997) Development of a fast and simple method for determination of beta-agonists in urine by extraction on empore membranes and detection by a test strip immunoassay. Journal of Agricultural and Food Chemistry. 45 (8), 3129–3137. Vulliet E., Baugros J.B., Flament-Waton M.M., GrenierLoustalot M.F. (2007) Analytical methods for the determination of selected steroid sex hormones

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and corticosteroids in wastewater. Analytical and Bioanalytical Chemistry. 387 (6), 2143–2151. Wang J., Dong M.H., Shimb W.J., Kannan N., Li D.H. (2007) Improved cleanup technique for gas chromatographic mass spectrometric determination of alkylphenols from biota extract. Journal of Chromatography A. 1171 (1–2), 15–21. Wang S., Huang W., Fang G.Z., Zhang Y., Qiao H. (2008) Analysis of steroidal estrogen residues in food and environmental samples. International Journal of Environmental and Analytical Chemistry. 88 (1), 1–25. Weltin D., Gehring M., Tennhardt L., Vogel D., Bilitewski B. (2004) Analytical workshop on endocrine disruptors. Beitrage zu Abfallwirts-chaft and Altlasten. Schriftenreihe des Institutes fur Abfallwirtschaft and Altlasten, TU Dresden 18, 35.

Williams R.J., Johnson A.C., Smith J.J.L., Kanda R. (2003) Steroid estrogens profiles along river stretches arising from sewage treatment works discharges. Environmental Science and Technology. 37 (9), 1744–1750. Zacur H.A., Linkins S., Chang V., Smith B., Kimball A.W., Burkman R. (1991) Ethinyl estradiol and norethindrone radioimmunoassay following Sephadex LH-20 column chromatography. Clinica Chimica Acta. 204 (1–3), 209–215. Zherdev A.V., Dzantiev B.B., Trubaceva J.N. (1997) Homogeneous enzyme immunoassay for pyrethroid pesticides and their derivatives using bacillary alphaamylase as labeled. Analytica Chimica Acta. 347 (1– 2), 131–138.

Chapter 10 Phthalates Jiping Zhu, Rong Wang, Yong-lai Feng, and Xu-liang Cao

Introduction Phthalates, also known as phthalic diesters, are chemicals that share a common structure feature of 1,2-benzenedicarboxylic acid (Figure 10.1). The differences among various phthalates are the structures of the two hydrocarbon chains (R1 and R2) attached to the two carboxylic acid functional groups. The smallest chain in phthalates is the methyl group forming dimethyl phthalate (R1 = R2 = −CH3, DMP). Longer chains such as C12 (di-dedecyl phthalate) and C13 (di-tridecyl phthalate) have also been reported. Because of the large differences in the length of the side chains, wide ranges of physical properties such as vapor pressure and octanol–water partition coefficient are observed for phthalates (Cousins and Mackay 2000). Some of these physical properties are summarized in Table 10.1 for the six most commonly monitored phthalates. Toxicology and epidemiology studies indicate that in animals and humans some phthalates can mimic hormones and have endocrine-disrupting properties. For example, studies using mammalian estrogens screening in vitro reveal that butyl benzyl phthalate (BBzP) and di-n-butyl phthalate (DBP) are weak estrogenics (Jobling et al. 1995). Studies on rodents have shown that phthalates are estrogenic and are associated with adverse Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

reproductive effects (Higuchi et al. 2003; Fukuwatari et al. 2002; Lamb et al. 1987; Poon et al. 1997). Significantly high levels of phthalates, including di-(2-ethylhexyl) phthalate (DEHP) and its metabolite, were observed in the samples of very young Puerto Rican girls with premature thelarche (Colón et al. 2000). There was an inverse linear association between the levels of some phthalate metabolites (monobutyl phthalate and monobenzyl phthalate) in urine and observed mobility, concentration, and normal morphology of sperm in American men (Duty et al. 2003). Phthalates are widely used in modern society. For example, DEHP is added to polyvinyl chloride (PVC), a thermoplastic polymer, to make it softer and more flexible. DEHP is also used in food-packaging materials, medical products such as intravenous tubing, plastic toys, vinyl upholstery, shower curtains, adhesives, and coatings. Phthalates with smaller side chains such as diethyl phthalate (DEP) and DBP are used as solvents in perfumes. Phthalates have been reported to be present in the environment, including water and soil (do Nascimento Filho et al. 2003; Peñalver et al. 2000; Fauser and Thomsen 2002; Suzuki et al. 2001; Luke-Betlej et al. 2001), consumer products (Aurela et al. 1999; Uhde et al. 2001; Wilkinson and Lamb 1999; Bouma and Schakel 2002), medical devices (Inoue et al. 2003), marine ecosystems (Lin et al. 2003), indoor air (Otake et al. 2001; Zhu et al. 2003), and indoor dust (Rudel et al. 2003; Butte and 255

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Analysis of Endocrine Disrupting Compounds in Food

Table 10.1. Physical properties of six commonly monitored phthalates. Name

Abbreviation CAS No. Structural formula Molecular weight Melting point (°C) Boiling point (°C) VP(Pa) (25°C) Specific gravity (20°C) Log KOW (25°C)* Log KOA (25°C)* Log KAW (25°C)*

Dimethyl Phthalate

Diethyl Phthalate

Di-n-butyl Phthalate

Butyl Benzyl Phthalate

Di(2ethylhexyl) Phthalate

Di(n-octyl) Phthalate

DMP 131-11-3 C10H10O4

DEP 84-66-2 C12H14O4

DnBP 84-74-2 C16H22O4

BBzP 85-68-7 C19H20O4

DEHP 117-81-7 C24H38O4

DnOP 117-84-0 C24H38O4

194.2

222.2

278.4

312.4

390.6

390.6

−40

−35

−35

−47

−25

282

298

340

370

385

428

2.63 × 10−1

2.83 × 10−3

2.17 × 10−4

1.17 × 10−4

1.19 × 10−6

1.00 × 10−7

1.192

1.118

1.042

1.111

0.986

0.978

1.61

2.54

4.27

4.7

7.73

9.46

7.01

7.55

8.54

8.78

10.53

11.52

−5.01

−4.27

−4.08

−2.8

−2.06

5.5

−5.4

* Values cited from Cousins and Mackay (2000).

O OR1 OR2 O Figure 10.1. General structure of phthalates.

Heinzow 2002). Phthalates are also widely present in dairy food (Zhu et al. 2010) and food in general (Cao 2010). The presence of phthalates in food is attributed to the contamination of phthalates from both food processing and migration of phthalates from food packaging materials into food (Cao 2010). Adipates such as di-2-ethylhexyl adipate (DEHA) have a core structure of 1,6-hexadicarboxylic acid (—HOCOC4 H8COOH—) instead of 1,2-benzenedicarboxylic acid (—HOCOC6H4COOH—) in the molecule. Adipate, DEHA in particular, is used in many food-processing products, including food wraps, and is often detected in food (Page and Lacroix 1992). Because

DEHA was often comeasured with phthalates, it is included in this chapter as well. Food is a general term for anything that is edible for humans. For the discussion of analytical methods in this chapter, we have grouped food into (a) water, including bottled water; (b) beverages and wines; (c) oils; (d) other nonfatty foods such as fruits, vegetables, and cereal grains; (e) milk; (f) other milk products, including cheese, cream, butter, and yogurt; and (g) other solid foods in general that are not covered in the above mentioned groups, including animal tissues and fat, such as meat or fish products, and cooked meal, such as duplicate/total diet samples and baby food (Table 10.2). Because of the complex nature of food matrices, analysis of food samples for phthalates involves sample preparation and extraction, further cleanup, in some cases, and instrumental separation and detection. Extraction and cleanup are the most challenging parts in the analysis of phthalates in foods and are often the critical steps in deciding

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SPME. Fibre: PDMS-DVB. Extraction time: 60 min. Extraction temperature: 25°C Stir bar (coated with PDMS) sorptive extraction followed by desorption of phthalates from stir bar by organic solvents. SPE. Eluted with 5 mL of dichloromethane/ hexane (4:1) and 5 mL of methanol/ dichloromethane (9:1) at 1 mL/min. SPME. Fibre: PDMS-DVB. Extraction time: 20 min. Extraction temperature: 25°C HS-SPME. Fibre: PDMS-DVB. Extraction time: 60 min. Extraction temperature: 90°C.

Drinking water

Drinking water

Bottled water

Wines

Beverages

b. Beverage and wine Beverages

Bottled water

LLE. Extraction with hexanedichloromethane (10:1), twice. Extract reconstituted to acetonitrile for analysis. Simultaneous steam distillation and extraction. HS-SPME. Fibre: PDMS-DVB. Extraction time: 60 min. Extraction temperature: 70°C.

SPME. Fibre: PDMS-DVB. Extraction time: 20 min. Extraction temperature: 100°C

Water

Bottled water

SPME. Fibre: PDMS-DVB. Extraction time: 30 min. Extraction temperature: 80°C

SPME. Fibre: polyacrylate. Extraction time: 90 min. Extraction temperature: 45°C

Water

a. Water Water

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

n/a

Cleanup

Table 10.2. Summary of sample preparation and cleanup methods reported in the literature.

GC-MS

GC-MS

HPLC-UV

GC-MS

GC-MS

GC-MS

GC-MS

GC-MS

GC-MS

GC-MS

GC-MS

Analysis

DEP, DBP, BBzP, DEHP, DEHA DMP, DEP, DBP, DEHP, BBzP, DOP

DBP, DEHP

DMP, DEP, DiBP, DBP, DEHP DMP, DEP, DiBP, DBP, BBzP, DHP, DEHA, DEHP, DOP

DMP, DEP, DBP, BBzP, DEHP

DMP, DEP, DBP, BBzP, DEHA, DEHP, DOP

DMP, DEP, DBP, BBzP, DEHA, DEHP, DOP DMP, DEP, DBP, BBzP, DEHA, DEHP, DOP DMP, DEP, DBP, BBzP, DEHP, DOP DEP, DBP, BBzP, DEHP

Analytes

Luke-Betlej et al. 2001 Serodio and Nogueira 2006 Casajuana and Lacorte 2003 Montuori et al. 2008 Cao 2008

5–40 μg/L

2–4 μg/L

(continued)

Kato et al. 2002 Carrillo et al. 2007

2–5 μg/L 150–2200 μg/L

Yano et al. 2002

4 mg/kg

20 μg/L for DEHP 3–85 μg/L

3–40 ng/L

Polo et al. 2005

Peñalver et al. 2001

Peñalver et al. 2000

Reference

2–103 ng/L

2–27 ng/L

7–170 μg/L

Detection Limit

258

Milk

Fruits of Benincasa hispida (e) Milk Milk

GPC with a Bio-Beads SX3 column. Sample eluted with dichloromethane-cyclohexane (1 : 1).

GPC with a PL-gel column. Sample eluted with pentaneMTBE (1 : 1).

LLE. Milk added with sodium chloride and water. Extracted with pentane-acetone followed by hexane-MTBE. Acetone washed away with water. LLE. Milk added with potassium hydroxide. Extracted with methanol-hexane (5:3). Centrifuged for phase separation. Organic layer reconstituted to dichlormethanecyclohexane (1:1) for cleanup.

GC-MS

GC-MS

GC-MS

GC-FID

n/a

Column cleanup with a silica gel column. Eluted with hexane-chloroform.

GC-MS

GC-ECD

GC-FID

GC-MS

Analysis

n/a

n/a

n/a

n/a

Cleanup

SLE. Powdered sample extracted with chloroform.

LLE or SLE. Sample blended with acetonitrile and filtered. Filtrate diluted with water and extracted with hexane and dichloromethane (10:1). Organic layer collected, washed with water, dried through sodium sulphate, evaporated to dryness, and redissolved in hexane for analysis. LLE or SLE. Sample extracted with heptane.

HS-SPME. Fibre: PDMS. Extraction time: 60 min. Extraction temperature: 40°C.

Vegetable oil

d. Other non-fatty food Non-fatty foods (fruits, vegetables, fruit juices and drinks, wines, beers, maple syrup, cereal grains) Fruit juices, soft drinks, fruit

Sample (4 g) dissolved in heptane (20 mL) and analysed directly by GC.

Samples analyzed directly without extraction and cleanup.

Edible oil

c. Oil Citrus essential oils

Table 10.2. Summary of sample preparation and cleanup methods reported in the literature. (cont.)

DEHP

DEP, DBP, BBzP, DEHP, DOP

DEHP

DEHP, DiOP, DEHA

DEP, DiBP, DBP, BBzP, DEHP, DOP, DEHA

DMP, DEP, DBP, BBzP, DEHP, DOP

DMP, DEP, DPP, DBP, DiBP, BBzP, DEHP, DOP DEHP, DiOP, DEHA

Analytes

Castle et al. 1990

Hogberg et al. 2008

0.12–3.0 μg/L

5 mg/kg

Kozyrod and Ziaziaris 1989 Du et al. 2006

Page and Lacroix 1995

Kozyrod and Ziaziaris 1989 Holadova et al. 2007

di Bella et al. 1999

Reference

30–70 mg/kg

50–500 mg/kg

0.2–0.5 mg/kg

30–70 mg/kg

3–40 μg/L

Detection Limit

259

Powdered infant formula

Milk and cream

(f) Milk products Milk, cream, butter, cheese SLE. Sample mixed with methanol-hexane and potassium hydroxide, shaken and centrifuged. The organic phase collected and the lower phase extracted twice with hexane. The combined hexane extracts evaporated and redissolved in dichloromethane-cyclohexane (1:1) for cleanup. SLE. Sample blended with acetone and hexane and centrifuged. Hexane removed and extraction with hexane repeated. Hexane extract dried and made to 5 mL of hexane. SLE. Sample mixed with hexane-saturated acetonitrile, sonicted, and centrifuged. Sample filtered and organic solution washed with hexane saturated with acetonitrile-saturated hexane, and volume reduced for analysis.

HS-SPME. Fibre: PDMS. Extraction time: 60 min. Extraction temperature: 90°C.

Cow milk and human milk

Milk

LLE. Extracted with ethanol-diethyl ether-pentane (2:1:1), followed by diethylether-pentane (1:1). Organic phase washed with 2% NaCl solution, dried, and redissolved in dichloromethanecyclohexane (1:1) for cleanup. LLE. Extracted twice with methanolhexane-MTBE (1.5:2:2). Organic phase dried, evaporated and redissolved hexane for cleanup.

Milk

n/a

Phthalates isolated from lipid by sweep codistillation, Florisil trapping, and selective elution.

GPC with a Bio-Beads SX3 column. Eluted with dichloromethane-cyclohexane (1 : 1).

a. For DBP, BBzP, and DEHP, the extract was mixed with acetonitrile. Hexane phase was discarded, and acetonitrile phase washed with hexane. b. For DiNP and DiDP, extract was loaded to a deactivated silica column and eluted with 0.7% ethyl acetate in hexane. n/a

GPC with a Bio-Beads SX3 column. Sample eluted with dichloromethane-cyclohexane (1 : 1). The eluate changed to isooctane for analysis.

Cleanup

Analysis

GC-MS

GC-MS

GC-MS

GC-MS

LC/MS/ MS

GC-ECD

Analytes

DBP, DEHP

DEP, DiBP, DBP, BBzP, DEHP, DOP, DEHA

DEHP

DMP, DEP, DBP, BBzP, DEHP, DOP

DBP, BBzP, DEHP, DiNP, DiDP

DEHP

0.05–0.5 mg/g

(continued)

Yano et al. 2005

Page and Lacroix 1995

Sharman et al. 1994

Feng et al. 2005; Zhu et al. 2006

0.12–1.8 μg/kg for 3.25% milk

0.01 mg/kg

Soerensen 2006

4–9 μg/kg

Reference Petersen 1991

0.05–0.2 mg/L

Detection Limit

260

Candy, chocolate, meat pasty, pork pie, chicken pie, sandwiches Cheese, meats, fish, sandwiches, cakes, fruit, vegetables, cooked meals Duplicate diet samples

g. Other solid food Fatty foods (animal tissues, fats, cheese) Baby food, infant formulae, total diet samples SLE. Sample extracted twice with acetone-hexane (1:1). The extract dried with sodium sulphate, evaporated to dryness, and redissolved in dichloromethane-cyclohexane (1:1) for cleanup. SLE. Sample blended and extracted with acetone-hexane (1:1). The extract dried over sodium sulphate, evaporated to dryness, and the residue redissolved in dichloromethane-cyclohexane (1:1) for cleanup. SLE. Sample extracted with acetonitrile twice. Acetonitrile layer mixed with sodium chloride and extracted with acetonitrile-saturated hexane. Acetonitrile collected, evaporated, and redissolved in hexane.

SLE. Sample blended with sodium sulphate and dicloromethane and filtered. Dichloromethane was chaned to 5 mL hexane for cleanup. SLE. Sample extracted with pentane. Supernatant reduced to dryness. Residue dissolved in 5 mL ethylacetatecyclohexane (1:1) for cleanup. GC-MS

GPC with a Bio-Beads SX3 column. Eluted with ethyl acetate-cyclohexane (1 : 1). Elutate fraction reduced to dryness and redissolved in 0.5 mL isooctane. GPC with a Bio-Beads SX3 column. Eluted with dichloromethane-cyclohexane (1 : 1). GC-MS

GC-MS

GPC with a Bio-Beads SX3 column. Eluted with dichloromethane-cyclohexane (1 : 1). Column cleanup with a Florisil and Bondesil PSA dual layer column, eluted with 5% of acetone in hexane. Eluate evaporated and redissolved in hexane.

GC-MS

GC-MS

Analysis

Phthalates isolated from lipid by sweep codistillation, Florisil trapping, and selective elution.

Cleanup

Table 10.2. Summary of sample preparation and cleanup methods reported in the literature. (cont.)

DEP, DPP, DBP, DPeP, DHP, BBzP, DCHP, DEHP, DiOP, DOP, DiNP, DEHA

DEHA

DBP, DCHP, BBzP

DBP, BBzP, DEHP, DEHA

DEP, DiBP, DBP, BBzP, DEHP, DOP, DEHA

Analytes

Castle et al. 1988a, b

Startin et al. 1987

Tsumura et al. 2001, 2003

<0.1 mg/kg

0.2–25.8 μg/kg

Petersen and Breindahl 2000

Page and Lacroix 1995

Reference

0.05 mg/kg

0.015–0.35 mg/ kg

0.05–0.5 mg/g

Detection Limit

Phthalates

the levels of detection limits of the overall methods.

Contamination issues in the analysis of phthalates Phthalates are common contaminants in the laboratory environment. They are present in air, and are found in many solvents; reagents; and common laboratory materials such as tubing, cork, glass wool, filter paper, alumina, Florisil, sodium sulfate, sodium chloride, and so on. The potential contamination of phthalates during sample preparation and instrument analysis has been mentioned in a number of studies. Among the phthalates, DEHP is usually the predominant one found in the blank, whereas DBP and DEP are also commonly present. Every precaution must be taken to minimize contamination and to achieve a low and stable blank level. The common precautions included rinsing all glassware coming into contact with samples using organic solvents such as methanol, acetone, and hexane (Soerensen 2006; Sharman et al. 1994; Page and Lacroix 1995; Petersen and Breindahl 2000); heating the glassware to a high temperature (Yano et al. 2005; Bruns-Weller and Pfordt 2000); or using a combination of heat treatment followed by solvent rinse (Gruber et al. 1998; Tsumura et al. 2001). In addition, septa, caps, and sample vials (Sharman et al. 1994); glass wool (Tsumura et al. 2001); and utensils for sample preparation (Page and Lacroix 1995) were generally rinsed with solvent before use. Other measures to reduce phthalate contamination included careful selection and prescreening of solvents (Soerensen 2006), distillation of purchased solvents in an allglass device (Page and Lacroix 1992, 1995) or purification with aluminum oxide (Fankhauser-Noti and Grob 2007), and avoidance of latex or vinyl gloves when handling samples (Page and Lacroix 1992). When adsorbent is to be used in sample cleanup, it should be decontaminated by heating prior to use (Page and Lacroix 1992; Soerensen

261

2006). In some cases, sodium chloride for sample handling was heat treated prior to use as well (Tsumura et al. 2001).

Sample pretreatment, extraction, and cleanup Water Water is a relatively simply matrix among foods. Solid-phase microextraction (SPME) has been widely used in the extraction of phthalates from water (Peñalver et al. 2000, 2001; Polo et al. 2005), drinking water (LukeBetlej et al. 2001), and bottled water (Montuori et al. 2008). SPME is a solventfree extraction method developed by Arthur and Pawliszyn in the early 1990s (1990). In SPME, a polymer-coated fiber is immersed in the water, and the target analytes are extracted onto the fiber. This is also often referred to as direct SPME because the fiber is in direct contact with the sample matrix. Because the water molecule is polar and has a minimal partition capability in the fiber selected for extracting nonpolar and lipophilic compounds such as phthalates, the target phthalates can therefore be concentrated effectively from water onto the fiber with the right choice of SPME fiber. PolydimethylsiloxaneCarbowax-divinylbenzene-coated (PDMSDVB-coated) fiber was reported to be the most common. The extraction temperature ranged from 25°C to 100°C, with various extraction times among various studies. The exposed fiber is inserted immediately after SPME into a gas chromatograpy/mass spectrometry (GC/MS) instrument for analysis without a need for further cleanup. One of the advantages of the SPME method is the elimination of the use of organic solvents, which often are contaminated with phthalates to some degree, and thus achieving low blank level and good method detection limits at nanograms per liter (Peñalver et al. 2001; Polo et al. 2005) to micrograms per liter (Peñalver et al. 2000; Luke-Betlej et al. 2001; Montuori et al. 2008) (Table 10.2).

262

Analysis of Endocrine Disrupting Compounds in Food

Besides direct SPME, headspace (HS) SPME was also used for the measurements of phthalates in bottled water, with the detection limit at low micrograms per liter (Cao 2008). In HS-SPME, the fiber is suspended right above the sample in a closed system, such as a vial, instead of being immersed in the sample. Because phthalates are semivolatile compounds with low vapor pressures (Table 10.1), the liquid sample usually is heated to aid the release of phthalates into the headspace where the extraction occurs. The advantage of HS-SPME is its ability to eliminate the coextraction of interferences, such as nonvolatile, large molecules in a complex matrix that are not suitable for gas chromatographic analysis (Feng et al. 2005). The other methods reported for the measurements of phthalates in water included stir bar sorptive extraction followed by liquid desorption (Serodio and Nogueira 2006) and traditional solid-phase extraction (SPE) methods (Casajuana and Lacorte 2003).

Beverages and wine Three different extraction methods have been reported for the measurements of phthalates in beverages and wine. They include liquid– liquid extraction (LLE) with hexanedichloromethane (DCM) (Yano et al. 2002), simultaneous steam distillation and extraction (Kato et al. 2002), and HS-SPME (Carrillo et al. 2007). All samples were analyzed without further cleanup. The extract from LLE was concentrated and reconstituted to acetonitrile (ACN) for the analysis by high-performance liquid chromatography (HPLC). The method detection limit, at the milligram per kilogram level in this method (Yano et al. 2002), was higher than the other two methods, likely due to the use of a UV detector instead of a MS detector (see Instrument Analysis below).

Fruit and vegetable oils Oils were analyzed either directly without any sample treatment (di Bella et al. 1999) or

diluted in heptane prior to GC analysis (Kozyrod and Ziaziaris 1989). Recently, the HS-SPME method was developed for the analysis of phthalates in vegetable oil (Holadova et al. 2007). An unmodified PDMS fiber instead of PDMS/PVB fiber was used, and the extraction was conducted at a lower temperature of 40°C. The high detection limits of milligrams per kilogram in the latter two cases were largely attributed to the use of a FID (flame ionization detector) (Kozyrod and Ziaziaris 1989) or ECD (electron capture detector) (Holadova et al. 2007) instead of using MS as a detector.

Other nonfatty food Only a few studies on the measurements of phthalates in solid, nonfatty food have been reported. Solvent extraction method, either LLE or solid–liquid extraction (SLE), depending on the type of foods, was employed using heptane (Kozyrod and Ziaziaris 1989) or chloroform (Du et al. 2006) as the extraction solvent to extract phthalates from various nonfatty foods including soft drinks, fruits, and fruit juice. Page and Lacroix employed ACN for the initial extraction of phthalates from various nonfatty foods, both liquid and solid, and the ACN extract was further extracted with other water-insoluble organic solvents of hexane and dichloromethane (DCM) (1995). High detection limits of several dozen (Kozyrod and Ziaziaris 1989) to several hundred (Page and Lacroix 1995) milligrams per kilogram were reported for these nonfatty foods.

Milk There are a number of reports on the measurements of phthalates in milk. Powdered milk sometimes was reconstituted by mixing it with water according to the manufacturer ’s preparation instructions. For example, Casajuana and Lacorte reported mixing milk

Phthalates

powder with water in a 1 : 10 ratio (3 g of powdered milk in 30 mL of water) prior to sample extraction (Casajuana and Lacorte 2004). Several analytical methods have been developed for the analysis of phthalates in milk as well. LLE using different combinations of organic solvents was commonly employed for the extraction of phthalates from milk. Because of the water content in the milk sample, generally a mixture of watersoluble solvents (methanol, ACN) and water-insoluble solvents (hexane, DCM, cyclohexane) was used for the initial extraction (Soerensen 2006; Hogberg et al. 2008; Castle et al. 1990; Petersen 1991). Such organic solvent systems included pentaneacetone followed by hexane-MTBE (methyl t-butyl ether) (Hogberg et al. 2008), methanolhexane in the presence of potassium hydroxide (Castle et al. 1990), ethanol-diethyl ether-pentane (Petersen 1991), and methanolhexane-MTBE (Soerensen 2006). Sodium chloride was added sometimes to aid the phase separation (Hogberg et al. 2008; Petersen 1991). The HS-SPME extraction method was used for the extraction of phthalates from raw milk (Feng et al. 2005) and human milk (Zhu et al. 2006). The method was first reported for the measurement of raw cow milk to study the effects of using phthalatecontaining tubing during the milking process in dairy farms on the phthalate levels in raw milk (Feng et al. 2005). Milk samples were mixed with sodium chloride and heated during HS-SPME extraction to help the release of phthalates from milk into the headspace. Although the absolute extraction efficiency for phthalates from milk due to high fat content was not as good as from water samples (Feng et al. 2005), the method detection limit of the HS-SPME method was reported to be at sub-micrograms per liter to low micrograms per liter, which is better than or comparable to LLE methods (Table 10.2).

263

Milk products Milk products such as cream, cheese, butter, and milk powder are solid materials and therefore SLE methods were often used by blending the samples with extraction solvents. Such solvent systems included methanol-hexane in the presence of potassium hydroxide (Sharman et al. 1994), acetone-hexane (Page and Lacroix 1995), and ACN-hexane (Petersen and Breindahl 2000). These methods were also applied to milk samples (Sharman et al. 1994; Page and Lacroix 1995). Because of the solid content in the extraction mixture, a centrifuge was used to aid the phase separation and collection of organic extracts. Detection limits of sub-milligrams per kilogram could be achieved using these methods.

Other solid food in general Besides milk and milk products, phthalates also have been measured in other solid foods in various studies involving duplicate diet and total diet samples. These samples contain various degrees of solid and fat content. Again, SLE with different solvent systems is the most common approach to extract phthalate from food into organic solvents. The organic solvent systems used in SLE include pentane (Petersen and Breindahl 2000) and acetone-hexane (Castle et al. 1988; Castle et al. 1988a, b). Tsumura et al. (2001, 2003) used ACN only for the initial extraction followed by LLE to extract phthalates from ACN into hexane for the duplicate diet samples. For some fatty foods such as animal tissues, fats, and cheese, samples were mixed with sodium sulfate and extracted with DCM. DCM was then collected through filtration for further cleanup (Page and Lacroix 1995). The detection limit for these solid foods was reported at sub-milligrams per kilogram.

Cleanup of extracts Once the initial extraction is completed, the extracts may or may not be subjected to

264

Analysis of Endocrine Disrupting Compounds in Food

further cleanup depending on the types of food. In general, nonfatty liquid food such as water, juice, beverages, and wine did not go through further cleanup procedures; they were analyzed once the phthalates were extracted either through SPME or LLE. The only exception was the employment of a cleanup procedure using a silica gel column for fruit samples as reported by Du et al. (2006). Oil was analyzed either directly (di Bella et al. 1999) or diluted (Kozyrod and Ziaziaris 1989) in organic solvents or using HS-SPME (Holadova et al. 2007) without further cleanup (Table 10.2). Further cleanup of the extracts is, however, almost always necessary for foods such as milk, milk products, or solid foods containing various degrees of fat and solid content because the matrices of these foods are much more complex. Isolation of phthalates from fat in fatty foods was often performed by sizeexclusion chromatography (SEC). Bio-Beads SX3 was the most commonly used SEC column, with DCM-cyclohexane as the eluting solvent (Sharman et al. 1994; Castle et al. 1990; Castle et al. 1988a, b; Startin et al. 1987), and the ethylacetate-cyclohexane system was also mentioned (Petersen and Breindahl 2000). The other SEC column reported was PL-Gel column with pentaneMTBE as the eluting solvents (Hogberg et al. 2008). Other, less popular approaches for cleanup of the extracts were to isolate phthalates by employing columns packed with silica gel (Soerensen 2006), Florisil (Page and Lacroix 1995), or a dual layer column of Florisil-Bondesil PSA (Tsumura et al. 2001, 2003).

Instrument analysis Compared to the challenges in extraction and cleanup of food samples, instrument analysis of the sample extracts is relatively simple. Because of the semivolatile, nonpolar nature of phthalates, instruments used in the analysis were mostly gas chromatography (GC), and HPLC-mass spectrometer (MS) (Soerensen,

2006) or HPLC-UV (Yano et al. 2002) were also employed. For GC analysis today, MS is almost a nominal detector for the measurement of phthalates, and other detectors, such as flame ionization detector (Kozyrod and Ziaziaris 1989) and the electron capture detector (Holadova et al. 2007; Petersen 1991), are also used.

Gas chromatography-mass spectrometry conditions For GC-MS analysis, prepared samples were injected into the GC-MS at an injection temperature around 250°C (240°C to 260°C reported from several studies). The commonly used GC capillary column for the separation of phthalates is a fused-silica capillary column containing 5% phenyl and 95% methyl polysiloxane with a column length of either 30 or 60 m. There are several different brands of such columns available, including HP-5, DB-5, ZB-5, XTI-5, etc. The oven temperature programs varied greatly depending on the complexity of the samples and number of target analytes monitored. For example, the rise of oven temperature ranged from 6°C/ min in one study to 15°C/min in another. Usually, the phthalates can be well separated under these oven temperature programs. The elution orders of commonly monitored phthalates and DEHA are listed in Table 10.3, along with the target ions (T-ion) for quantification and several characteristic ions for identification purposes. The separated phthalates can be detected by a mass spectrometer that is operated either in full-scan mode or in selected ion monitoring (SIM) mode. Mass spectra generated through electron impact (EI) ionization of the six most commonly monitored phthalates are presented in Figure 10.2. For phthalates, the base peak of the mass spectrum is always ion m/z 149, which is also served as the quantifying ion, except for DMP, which has a base peak of ion m/z 163. For DEHA, the base peak is ion m/z 129 (Table 10.3).

Table 10.3. Gas chromatography retention times of some commonly measured phthalates along with quantifying ions and characteristic ions. Abbr.

Full Name

DMP DEP DIBP DBP DPeP DNHP BBP DEHA DCHP DEHP DOP DDcP DUP

Dimethyl phthalate Diethyl phthalate Diisobutyl phthalate Di-n-Butyl phthalate Dipentyl phthalate Di-n-hexylphthalate Butyl benzyl phthalate Bis(2-ethylhexyl) adipate Dicyclohexyl phthalate Bis(2-ethylhexyl) phthalate Dioctyl phthalate Didecyl phthalate Diundecyl phthalate

M.W.

RT (min)

Q-Ion

Characteristic Ions

14.34 15.5 17.66 18.47 20.16 21.94 22.05 22.42 23.63 23.71 25.21 27.97 29.97

163 149 149 149 149 149 149 129 149 149 149 149 149

194 177 223, 167 223, 205 237 251 206, 91 259, 241, 147 167, 249 167, 279 279 307 321

194 222 278 278 306 334 312 370 330 390 390 446 474

Injection port temperature: 280°C; Column: DB-5MS 30 m × 0.25 mm I.D. × 0.25 μm film thickness; Oven temperature: 45°C (5 min), 15°C/min to 210°C, 8°C/min to 270°C, 30°C/min to 310°C (25 min). A b u n d a n c e

A b und a nce

S c a n 4 2 0 0 0 0

S c a n 1 6 2 6 ( 1 4 . 3 6 6 m i n ) : 0 8 0 8 0 8 1 1 . D \ d a ta . m s ( - 1 6 1 9 ) ( - ) 163

1 8 4 2 (1 5 .5 2 0 1 4 9

m i n ) : 0 8 0 8 0 8 1 1 . D \ d a ta . m s

(-1 8 3 8 ) (-)

4 0 0 0 0 0 3 8 0 0 0 0

400000

3 6 0 0 0 0 3 4 0 0 0 0

DMP

350000

3 2 0 0 0 0 3 0 0 0 0 0

DEP

2 8 0 0 0 0

300000

2 6 0 0 0 0 2 4 0 0 0 0

250000

2 2 0 0 0 0 2 0 0 0 0 0

200000 1 8 0 0 0 0 1 6 0 0 0 0

150000

1 4 0 0 0 0 1 2 0 0 0 0

100000

1 7 7

1 0 0 0 0 0 8 0 0 0 0

77 50000

6 0 0 0 0

194 50

104

133

4 0 0 0 0

0 40

60

1 0 5

7 6

2 2 1 2 4 1 2 5 9 2 8 23 0 03 1 7 3 3 73 5 5 3 7 6 3 9 6 4 1 5 4 3 6

2 0 0 0 0

80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440

m / z -->

0 2 0

5 0

2 2 2 1 9 5

1 2 3 4 0

6 0

8 0

2 4 3 2 6 2 2 8 1

3 1 4 3 3 13 4 9 3 6 9

4 0 1

4 2 6 4 4 6

1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0

m / z -->

A b u n d a n c e

A b und a nc e S c a n 2 4 0 1 ( 1 8 . 5 0 7 m i n ) : 0 8 0 8 0 8 1 1 . D \ d a ta . m s 1 4 9

(-2 3 9 4 ) (-)

S c a n 1 5 0 1 ( 1 6 . 0 1 0 m i n ) : 0 8 0 2 2 8 1 0 . D \ d a ta . m s ( - 1 4 9 4 ) ( - ) 1 49

4 00 000 0

6 0 0 0 0 0

5 5 0 0 0 0

3 50 000 0

BBzP

5 0 0 0 0 0

DBP

4 5 0 0 0 0

3 00 000 0

4 0 0 0 0 0

2 50 000 0 3 5 0 0 0 0

2 00 000 0

3 0 0 0 0 0

91 2 5 0 0 0 0

20 6

1 50 000 0 2 0 0 0 0 0

1 00 000 0

1 5 0 0 0 0

1 0 0 0 0 0

50 000 0 5 0 0 0 0 4 1

7 6

1 2 2

5 9

0 4 0

6 0

8 0

2 0 5

1 0 4 1 6 7 1 8 6

1 23 6 5

2 2 3

1 78

41 2 5 0

2 7 82 9 6

3 2 7

3 5 6 3 7 63 9 4

4 1 6

2 38 2 67

0

4 3 8

4 0

1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0

60

80

2 94

3 12

34 1

36 8

4 01

43 14 49

10 0 12 0 1 40 1 60 18 0 20 0 22 0 2 40 2 60 2 80 30 0 32 0 3 40 3 60 3 80 40 0 42 0 4 40

m / z -->

m / z --> A b u n d a n c e

A b u n d a n c e S c a n 3 3 8 4 (2 3 .7 5 8 1 4 9

3 4 0 0 0 0

m i n ) : 0 8 0 8 0 8 1 1 . D \ d a ta . m s

(-3 3 7 7 ) (-)

S c a n 3 6 6 0 ( 2 5 . 2 3 2 m i n ) : 0 8 0 8 0 8 1 1 . D \ d a ta . m s ( - 3 6 5 7 ) ( - ) 1 4 9

6 0 0 0 0 0

3 2 0 0 0 0

5 5 0 0 0 0

3 0 0 0 0 0

5 0 0 0 0 0

2 8 0 0 0 0

DEHP

2 6 0 0 0 0 2 4 0 0 0 0

DnOP

4 5 0 0 0 0

4 0 0 0 0 0

2 2 0 0 0 0

3 5 0 0 0 0

2 0 0 0 0 0 1 8 0 0 0 0

3 0 0 0 0 0 1 6 0 0 0 0

2 5 0 0 0 0

1 4 0 0 0 0 1 6 7

1 2 0 0 0 0

2 0 0 0 0 0

1 0 0 0 0 0

1 5 0 0 0 0 8 0 0 0 0 6 0 0 0 0

1 0 0 0 0 0

2 7 9

4 0 0 0 0

2 7 9

5 7

5 0 0 0 0 7 6 0

3 9 4 0

m / z -->

4 3

7 1

1 0 4

2 0 0 0 0

6 0

8 0

1 2 2

1 8 5 2 0 3 2 2 1 2 3 9

2 6 1

3 0 1 3 1 9

3 4 2 3 6 1

3 9 0

1 0 4 1 2 3

4 1 5 4 3 5

0 2 0

1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0

4 0

6 0

8 0

1 6 7

1 8 92 0 7

2 3 3

2 6 1

3 0 6

3 3 03 4 7 3 6 9 3 9 0 4 1 1 4 2 94 4 7

1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0

m / z -->

Figure 10.2. Electron impact mass spectra of six commonly monitored phthalates.

266

Analysis of Endocrine Disrupting Compounds in Food

O OR OR O - R + 2H

1

-OR

OH

O

OH OR

O

R

O

O

3 [M - OR]

2 [M - R + 2H]

-R+H

-R+H

OH OH OH

O - H2O

O

4 m/z = 167

OH O

206 and m/z 91 besides the base peak of ion m/z 149. Ion m/z 206 is formed by the loss of the neutral group of C6H5CHO, whereas the ion m/z 91 corresponds to a benzyl ion of [C6H5CH2]. The characteristic ions of phthalates resulting from the fragmentations under EI mode are summarized in Table 10.3. In practice, one has to consider the relative abundance of these fragments in selecting qualifier (Q-) ions. There were some variations in selecting Q-ions for phthalates among various studies. For example, Feng and others (2005) selected Q-ions of m/z 77 and 194 for DMP, m/z 177 and 104 for DEP, m/z 223 and 104 for DBP, m/z 91 and 206 for BBzP, m/z 167 and 279 for DEHP, and m/z 279 and 104 for DOP, whereas Casajuana and Lacorte (2004) selected Q-ions of 77 and 135 for DMP, m/z 177 and 105 for DEP, m/z 223 and 76 for DBP, m/z 91 and 206 for BBzP, and m/z 167 and 279 for DEHP.

5 m/z = 149

Figure 10.3. Possible fragmentation pathways of phthalates under electron impact ionization condition.

MS fragmentation pathways The fragmentation pathways of phthalates under EI ionization are illustrated in Figure 10.3. There are two fragmentation pathways, and both lead to the formation of an ion of m/z 149, except for DMP. DMP loses a methoxy group (—CH3O) under EI to form a stable ion of m/z 163. The ion of m/z 177 in the DEP spectrum is also formed through the loss of an ethoxy group. For phthalates with long side chains, such as DEHP, the major fragmentation pathway seems to be the loss of an alkyl side chain to form an (M − R + 2H) ion (m/z 279 in the spectrum of DEHP and DnOP). This ion is further fragmented to the ion of m/z 167, which ultimately loses H2O to form the stable ion of m/z 149. The mass spectrum of BBzP is dominated by ions m/z

Conclusion Although methods are available for the determination of phthalates in various food types, detection limits of these methods vary greatly depending on the method employed and the types of food. In general, the method detection limits for fatty foods are very high due to the high blank levels of phthalates caused by the contamination of laboratory reagents and various materials used. Thus, more stringent measures should be taken to minimize the blank levels of phthalates for existing methods in order to improve the detection limits. Efforts should also be made to develop methods that have the least number of steps and use the minimum amounts of solvents, adsorbents, and other laboratory supplies in order to minimize blank levels of phthalates. Methods for the existing list of phthalates should also be expanded to cover other plasticizers that have been used as replacements for DEHP and DEHA in some food-

Phthalates

packaging materials, such as di-iso-nonyl phthalate (DiNP), di-iso-decyl phthalate (DiDP), di-iso-nonyl adipate (DiNA), and (di(2-ethylhexyl)terephthalate (DEHT), in order to determine their presence in foods for human exposure assessment.

References Arthur, C.L. and Pawliszyn, J. (1990) Solid-phase microextraction with thermal desorption using fused silica optical fibers. Anal Chem. 62, 2145. Aurela, B., et al. (1999) Phthalates in paper and board packaging and their migration into Tenax and sugar. Food Addit Contamin. 16, 571. Bouma, K. and Schakel, D.J. (2002) Migration of phthalates from PVC toys into saliva simulant by dynamic extraction. Food Addit Contamin. 19, 602. Bruns-Weller, E. and Pfordt, J. (2000) Detemination of phthalic acid esters in foods, mother ’s milk, dust, and textiles. Umweltwissenschaften und SchadstoffForschung. 12, 125. Butte, W. and Heinzow, B. (2002) Pollutants in house dust as indicators of indoor contamination. Rev Environ Contam Toxicol. 175, 1. Cao, X.-L. (2008) Determination of phthalates and adipate in bottled water by headspace solid-phase microextraction and gas chromatography/mass spectrometry. J Chromatogr A. 1178, 231. Cao, X.-L. (2010) Phthalate esters in foods: Sources, occurrence, and analytical methods. Compr Rev Food Sci Food Saf. 9, 21. Carrillo, J.D., et al. (2007) Determination of phthalates in wine by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry: Fibre comparison and selection. J Chromatogr A. 1164, 248. Casajuana, N. and Lacorte, S. (2004) New methodology for the determination of phthalate esters, bisphenol A, bisphenol A diglycidyl ether, and nonylphenol in commercial whole milk samples. J Agric Food Chem. 52, 3702. Casajuana, N. and Lacorte, S. (2003) Presence and release of phthalic esters and other endocrine disrupting compounds in drinking water. Chromatogrgraphia. 57, 649. Castle, L., et al. (1990) Migration of plasticizer from poly(vinyl chloride) milk tubing. Food Addit Contam. 7, 591. Castle, L., et al. (1988a) Migration from plasticized films into foods. 4. Use of polymeric plasticizers and lower levels of di-(2-ethylhexyl)adipate plasticizer in PVC films to reduce migration into foods. Food Addit Contam. 5, 277. Castle, L., et al. (1988b) Migration from plasticized films into foods. 3. Migration of phthalate, sebacate, citrate, and phosphate esters from films used for retail food packaging. Food Addit Contam. 5, 9.

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Colón, I., et al. (2000) Identification of phthalate esters in the serum of young Puerto Rican girls with premature breast development. Environ Health Persp. 108, 895. Cousins, I. and Mackay, D. (2000) Correlating the physical-chemical properties of phthalate esters using the ‘three solubility’ approach. Chemosphere. 41, 1389. di Bella, G., et al. (1999) Contamination of Italian citrus essential oils: Presence of phthalate esters. J Agric Food Chem. 47, 1009. do Nascimento Filho, I., et al. (2003) Identification of some plasticizers compounds in landfill leachate. Chemosphere. 50, 657. Du, Q., et al. (2006) Di-2-ethylhexyl phthalate in the fruits of Benincasa hispida. Food Addit Contam. 23, 552. Duty, S.M., et al. (2003) Phthalate exposure and human semen parameters. Epidemiology. 14, 269. Fankhauser-Noti, A. and Grob, K. (2007) Blank problem in trace analysis of diethyhexyl and dibutyl phthalate: Investigation of the sources, tips, and tricks. Anal Chim Acta. 582(2), 353–360. Fauser, P. and Thomsen, M. (2002) Sensitivity analysis of calculated exposure concentrations and dissipation of DEHP in a topsoil compartment: The influence of the third phase effect and dissolved organic matter (DOM). Sci Total Environ. 296, 89. Feng, Y.-L., et al. (2005) Development of a headspace solid phase microextraction method combined with gas chromatography mass spectrometry for the determination of phthalate esters in cow milk. Anal Chim Acta. 538, 41. Fukuwatari, T., et al. (2002) Elucidation of the toxic mechanism of the plasticizers, phthalic acid esters, putative endocrine disrupters: Effects of dietary di(2ethylhexyl)phthalate on the metabolism of tryptophan to niacin in rats. Biosci Biotechnol Biochem. 66, 705. Gruber, L., et al. (1998) Analysis of phthalates in baby food. Deutsche Lebensmittel-Rundashau. 94, 177. Higuchi, T.T., et al. (2003) Effects of dibutyl phthalate in male rabbits following in utero, adolescent, or postpubertal exposure. Toxicol Sci. 72, 301. Hogberg, J., et al. (2008) Phthalate diesters and their metabolites in human breast milk, blood or serum, and urine as biomarkers of exposure in vulnerable populations. Environ Health Perspect. 116, 334. Holadova, K.G., et al. (2007) Headspace solid-phase microextraction of phthalic acid esters from vegetable oil employing solvent based matrix modification. Anal Chim Acta. 582, 24. Inoue, K., et al. (2003) The validation of columnswitching LC/MS as a high-throughput approach for direct analysis of di(2-ethylhexyl) phthalate released from PVC medical devices in intravenous solution. J Pharmaceut Biomed Anal. 31, 1145. Jobling, S., et al. (1995) A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ Health Persp. 103, 582. Kato, K., et al. (2002) Preparation of samples for gas chromatography/mass spectrometry analysis of

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phthalate and adipate esters in plasma and beverages by steam distillation and extraction. J AOAC Int. 85, 719. Kozyrod, R.P. and Ziaziaris, J. (1989) A survey of plasticizer migration into foods. J Food Prot. 52, 578. Lamb IV, J.C., et al. (1987) Reproductive effects of four phthalic acid esters in the mouse. Toxicol Appl Pharmacol. 88, 255. Lin, Z.-P., et al. (2003) Determination of phthalate ester congeners and mixtures by LC/ESI-MS in sediments and biota of an urbanized marine inlet. Environ Sci Technol. 37, 2100. Luke-Betlej, K., et al. (2001) Solid-phase microextraction of phthalates from water. J Chromatogr A. 938, 93. Montuori, P., et al. (2008) Assessing human exposure to phthalic acid and phthalate esters from mineral water stored in polyethylene terephthalate and glass bottles. Food Addit Contam. 25, 511. Otake, T., et al. (2001) Analysis of organic esters of plasticizer in indoor air by GC-MS and GC-FPD. Environ Sci Technol. 35, 3099. Page, B.D. and Lacroix, G.M. (1992) Studies into the transfer and migration of phthalate esters from aluminium foil paper laminates to butter and margarine. Food Addit Contam. 9, 197. Page, B.D. and Lacroix G.M. (1995) The occurrence of phthalate ester and di-2-ethylhexyl adipate plasticizers in Canadian packaging and food sampled in 1985– 1989: A survey. Food Addit Contam. 2, 129. Peñalver, A., et al. (2001) Comparison of different fibres for the solid-phase microextraction of phthalate esters from water. J Chromatogr A. 922, 377. Peñalver, A., et al. (2000) Determination of phthalate esters in water samples by solid-phase microextraction and gas chromatography with mass spectrometric detection. J Chromatogr A. 872, 191. Petersen, J. H. (1991) Survey of di-(2-ethylhexyl)phthalate plasticizer contamination of retail Danish milks. Food Addit Contam. 8, 701. Petersen, J.H. and Breindahl, T. (2000) Plasticizers in total diet samples, baby food, and infant formulae. Food Addit Contam. 17, 133. Polo, M., et al. (2005) Multivariate optimization of a solid-phase microextraction method for the analysis of phthalate esters in environmental waters. J Chromatogr A. 1072, 63. Poon, R., et al. (1997) Subchronic oral toxicity of di-noctyl phthalate and di(2-ethylhexyl) phthalate in the rat. Food Chem Toxicol. 35, 225. Rudel, R.A., et al. (2003) Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other

endocrine-disrupting compounds in indoor air and dust. Environ Sci Technol. 37, 4543. Serodio, P. and Nogueira, J.M.F. (2006) Consideration on ultra-trace analysis of phthalates in drinking water. Water Res. 40, 2572. Sharman, M., et al. (1994) Levels of di-(2-ethylhexyl) phthalate and total phthalate ester in milk, cream, butter, and cheese. Food Addit Contam. 11, 357. Soerensen, L. (2006) Determination of phthalate in milk and milk products by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 20, 1135. Startin, J.R., et al. (1987) Migration from plasticized films into foods. 1. Migration of di-(2-ethylhexyl) adipate from PVC films during home-use and microwave cooking. Food Addit Contam. 4, 385. Suzuki, T., et al. (2001) Monitoring of phthalic acid monoesters in river water by solid-phase extraction and GC-MS determination. Environ Sci Technol. 35, 3757. Tsumura, Y., et al. (2001) Eleven phthalate esters and di(2-ethylhexyl) adipate in one-week duplicate diet samples obtained from hospitals and their estimated daily intake. Food Addit Contam. 18, 449. Tsumura, Y., et al. (2003) Estimated daily intake of plasticizers in 1-week duplicate diet samples following regulation of DEHP-containing PVC gloves in Japan. Food Addit Contam. 20, 317. Uhde, E., et al. (2001) Phthalic esters in the indoor environment–test chamber studies on PVC-coated wallcoverings. Indoor Air. 11, 150. Wilkinson, C.F. and Lamb IV, J.C. (1999) The potential health effects of phthalate esters in children’s toys: A review and risk assessment. Regul Toxicol Pharmacol. 30, 140. Yano, K., et al. (2002) Phthalate levels in beverages in Japan and Korea. Bull Environ Contam Toxicol. 68, 463. Yano, K., et al. (2005) Phthalates levels in baby milk powder sold in several countries. Bull Environ Contam Toxicol. 74, 373. Zhu, J., et al. (2006) Phthalate esters in human milk: Concentration variations over a six-month postpartum time. Environ Sci Technol. 40, 5276. Zhu, J., et al. (2003) Phthalates in indoor air of Canadian residences. In: Proceedings of the ISIAQ 7th International Conference. Vol. 1, pp. 542–547. Zhu, J., et al. (2010) Chemical contaminants: Phthalates. In: Handbook of Dairy Foods Analysis. Nollet, L.M.L. and Toldrá, F., eds. CRC Press, Boca Raton, Florida, pp. 771–800.

Chapter 11 Organotin Compounds Analysis Maw-Rong Lee and Chung-Yu Chen

Introduction Organotin (OT) compounds were first used in polymer synthesis stabilizers in 1936. Since then, organotin compounds have been extensively used in glass, plastics, paint, paper, leathers, and in textile industries. In the 1950s, tri-substitute organotin compounds were found to have interactions with some organisms and have been used as fungicides, bactericides, pesticides, and biocides in agriculture since that time (Huang et al. 2004). However, organotin compounds are now major contaminants that are continuously released by industries, agricultural firms, and by antifouling agents in the paints of ship hulls. Moreover, organotin compounds are found to affect marine life as well as marine birds, mammals, and human beings through the food chain (de Mora et al. 2003; Dorneles et al. 2008). In 1997, Kannan and Falandysz, two environmental scientists, detected a measurable butyltin concentration in a human subject’s liver, presumably due to the subject’s seafood consumption (Kannan and Falandysz, 1997). Organotin compounds are toxic, persistent substances that are considered to be endocrinedisrupting chemicals (Lee et al. 2005). Tributyltin (TBT) and triphenyltin (TPhT), Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

among others, are common organotin compounds used in various fields. Tributyltin and triphenyltin are highly toxic to many aquatic species and especially to their sensitive endocrine systems even at low concentrations. Imposex has been known to occur in some mollusks living in waters with concentrations of 1 ng/L of tributyltin (Hoch 2001; Formme et al. 2005). In 1982, the usage of antifouling paints containing tributyltin on boats less than 25 m long was banned by French authorities (Formme et al. 2005). After 1989, the regulations on the usage of tributyltin-containing paints came into effect in the United States, Australia, New Zealand, South Africa, Japan, and most countries in Europe (Diez et al. 2005). In Taiwan, a complete ban of the agricultural use of triphenyltin was implemented in 1999. In 2003, the use of tributyltin as an antifouling agent on small boats (<25 m length) was prohibited by the U.S. EPA (Environmental Protection Agency) (Meng et al. 2009). Despite all this regulation, however, tributyltin can still be found in the environment because of its slow degradation rate and high absorption in suspended matter and sediment (Belfroid et al. 2000). In this chapter, we describe common sample preparation techniques for extracting organotin compounds from various matrices and detection methods for analyzing organotin compounds. We also discuss the applications of organotin compound analysis in 269

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seafood, seawater, sediment, sewage sludge, body fluids, and other solid matrices, including soil, textiles, and plastics.

Sample preparation and detection methods for organotin compound analysis Sample preparation for extracting organotin compounds For trace analysis in complex matrices, proper sample preparation determines the validation of analytical samples and results. Several sample preparation techniques including liquid–liquid extraction (LLE), purge and trap extraction, solid-phase extraction (SPE), accelerated solvent extraction (ASE), microwave-assisted extraction (MAE), solidphase microextraction (SPME), and liquidphase microextraction (LPME) were used to extract organotin compounds in various sample matrices. Liquid–liquid extraction Liquid–liquid extraction (LLE) is also referred to as immiscible solvent extraction due to the immiscibility of the liquid-phase sample and the extraction solvent. LLE is based on Nernst’s distribution law: the analyte is distributed between the two immiscible liquids according to the relative solubility in each solvent. In 1980, dichloromethane was used as the extraction solvent to extract the phenyltin species including triphenyl chloride, diphenyl dichloride, phenyltin trichloride, tetraphenyltin, and inorganic tin (Sn4+ plus SnO2) to demonstrate the degradation of triphenyltin hydroxide in water (Soderquist and Crosby 1980). Dichloromethane was also used to extract tributyltin in tissue and sediment and propylation derivatives of organotin compounds in starfish and bivalves (Stephenson and Smith 1988; Shim et al. 2005). The mix of methanol and dichloromethane solvent was used for extracting organotin compounds in salmon (Sullivan et al. 1988). For extracting organotin species

in sediment, dried mussel, and sewage sludge, methanol and glacial ethanoic acid were used as an extraction solvent. The extract was derivatized with sodium tetraethylborate (NaBEt4), and the organotin compound derivatives were extracted by iso-octane, then analyzed by gas chromatography (GC) (Montigny et al. 1998). Hexane and iso-octane were also used to extract the sodium tetraethylboratederivatized organotin compounds in water, sediment, and fish tissue (Looser et al. 2000; Encinar et al. 2001; Nikolaou et al. 2007; Tang and Wang 2007; Evans et al. 2009). Purge and trap extraction Purge and trap extraction, also called dynamic headspace extraction, is used for the extraction of volatile or semivolatile analytes in solid or liquid samples. The analytes are continuously removed from a sample matrix by a flowing inert gas such as nitrogen or helium. For organotin compound extraction, methyltin and butyltin compounds in aqueous samples are in situ derivatized with sodium tetraethylborate in an 800-mL sample, purged with helium gas, and then cryofocused at −40°C in the Tenax-filled glass insert of a modified split/splitless injector (Eiden et al. 1998). After thermal desorption, the derivatized tin species are refocused on the capillary GC column at 10°C subsequently by GC separation and mass spectrometric detection. The six organotin compounds in marine sediments are then leached with acetic acid/methanol (1 : 9) solution; derivatized with sodium tetraethylborate; then stripped by a flow of helium, preconcentrated with the trap coated with Tenax GC, silica gel, and activated carbon; and finally thermally desorbed (Campillo et al. 2004). The extract is followed by gas chromatography with microwave-induced plasma atomic emission spectrometry (GC-AED). Solid-phase extraction Solid-phase extraction is a robust method that offers advantages over conventional liquid– liquid extraction techniques by having a gen-

Organotin Compounds Analysis

erally higher recovery, lower consumption of organic solvents, and a fast extraction speed, which is mainly for laboratories providing large number of analyses with rapid reporting times. Solid-phase extraction is widely used for extraction of analytes in various matrix samples. Different cartridges of solid-phase extraction technique are used to extract organotin compounds in various matrices, including water, sediment, sewage sludge, and shellfish (Arnold et al. 1998; Mizuishi et al. 1998; González-Toledo et al. 2001, 2002; Muñoz et al. 2005). The most commonly used cartridge, C18, was used to extract triorganotin species in water samples. González-Toledo and his colleagues reported a 250-mL aliquot sample of water was loaded into the cartridge, and after retention, the cartridge was washed with 10 mL of water and then eluted with 2 mL of 0.02% trifluoroacetic acid in acetonitrilewater (50 : 50) (González-Toledo et al. 2002). The recoveries of organotin compounds for this extraction range from 80% to 110%. For extraction of organotin compounds in water, sediment, and sewage sludge samples, different cartridges, including graphitized carbon black (Carbopack B), Florisil, multiwalled carbon nanotubes (MWNTs), C60, and C70 are used as extraction sorbents (Arnold et al. 1998; Mizuishi et al. 1998; Muñoz et al. 2005). The limit of detection for these cartridges was at the picogram per milliliter level. González-Toledo and his colleagues also reported butyltin and phenyltin species in shellfish were extracted by ethyl acetate, and the ethyl acetate extract removed coextracted substances using a cleanup step based on a solid-phase extraction using a cation exchange-bonded (SCX-bonded) phase cartridge (González-Toledo et al. 2001). This method is applied to the analysis of diphenyltin, triphenyltin, and tributyltin in oyster and mussel tissues at the nanogram per gram level. Accelerated solvent extraction Accelerated solvent extraction, also known as pressurized fluid extraction (PFE) or pressur-

271

ized liquid extraction (PLE), uses solvents at high temperature (100°C to 180°C) and pressure (1500–2000 psi) to enhance the extraction efficiency of analytes from a sample of solid. The accelerated solvent extraction was evaluated to extract mono-, di-, and tributyltin in sediment by using methanol-acetic acid mixture as an extraction solvent (Encinar et al. 2002). The optimal extraction condition was an extraction time of 10 minutes at 110°C. At higher temperatures (140°C and 175°C), the decomposition reactions of organotin species were detected. The methanol-acetic acid mixture (85 : 15, v/v) was also used as extraction solvent to extract butyltin, phenyltin, and octyltin in soil samples at a constant temperature (100°C) and pressure (90 bars) (Heroult et al. 2008). This extraction method appears to be reliable for the determination of organotin compounds in soils. Microwave-assisted extraction Microwave-assisted extraction is based on the migration of ions and the rotation of dipoles. It has advantages in the reduction of extraction time and the increase of sample throughput. For extraction of organotin compounds in sediment, an acidic extraction solvent was used for leaching and extracting under a low-power focused microwave field (30–315 W). The extract subsequently was derivatized by sodium tetraethylborate, then extracted by organic solvent (Donard et al. 1995; Szpunar et al. 1996; Rodriguez et al. 1999; Tutschku et al. 2002). The tetramethylammonium hydroxide (TMAH) acidified by acetic acid was used to extract butyltin compounds in biomaterials and oysters (Szpunar et al. 1996; Monperrus et al. 2003). The extraction step was also under a lowpower microwave (60 and 40 W), and the extraction time was less than 5 minutes. For simultaneous extraction and derivatization of organotin compounds in fish tissues, the tissue sample, acetic acid, organic solvent (nonane), and derivatization agent (sodium

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tetraethylborate) were placed in an extraction tube, which was exposed to a microwave at the power of 40 W for 3 minutes (Pereiro et al. 1996). The organic supernatant was directly injected to gas chromatography with an atomic emission detector (GC-AED) for analysis. Solid-phase microextraction Solid-phase microextraction (SPME) is a solvent-free technique that is based on equilibrium of analytes between sample matrices and fused-silica fiber coated with specific stationary phase (Zhong et al. 1994). The analytes removed by extraction fiber are directly determined by thermal desorption into a gas chromatograph or desorption by an extraction solvent or the mobile phase of a highperformance liquid chromatograph. Several studies have addressed the SPME of water samples contaminated with organotin species (Lespes et al. 1998; Jiang et al. 2000; Millán and Pawliszyn 2000). Our lab developed in situ derivatization with sodium tetraethylborate and headspace adsorption on a SPME fiber coated with polydimethysiloxane (PDMS), then analyzed the results by gas chromatography–mass spectrometry (GC– MS) to determine trace levels of organotin compounds in seawater (Chou and Lee 2005). The derivatization and extraction device is shown in Figure 11.1. Under optimal extraction conditions, the detection limit is lower than the picogram per milliliter level, and the precision is less than 25%. The in situ derivatization-SPME adsorption method was also used to extract organotin species in alcoholic beverages (Liu and Jiang 2002; Heroult et al. 2008). For analysis of organotin compounds in solid samples, including sediment, soil, and fish tissue samples, acid leaching is a necessary step before derivatization and adsorption by SPME (Moens et al. 1997; Aguerre et al. 2003; Devos et al. 2005; Zuliani et al. 2006; Carvalho et al. 2007).

Figure 11.1. Schematic diagram of the extraction and derivatization device for organotin compounds determination in SPME.

Liquid-phase microextraction Liquid-phase microextraction is a novel extraction technique that extracts analyte in an aqueous sample by using small amounts of immiscible organic solvents. There are three types of liquid-phase microextraction techniques to extract organotin compounds in various matrices: single-drop microextraction, supported liquid membrane probe, and dispersive liquid–liquid microextraction. For single-drop microextraction, organic solvent was loaded into a gas-tight syringe and 1–2 μL of solvent was exposed in the water sample or extract from sediment or tissue matrices to extract organotin compounds by immersion or headspace (Shioji et al. 2004; Colombini et al. 2004; Xiao et al. 2008). The supported liquid membrane probe technique was introduced by Cukrowska et al. (2004). Iso-octane used as acceptor solvent was injected into a polypropylene tube and then immersed into samples of water to extract organotin compounds. In 2008 Birjandi et al. developed the dispersive

Organotin Compounds Analysis

liquid–liquid microextraction method to extract butyltin and phenyltin by mixing immiscible extraction solvent (carbon tetrachloride) and a sample of water in a conical test tube (Birjandi et al. 2008). After centrifugation, the carbon tetrachloride was collected in the bottom of the conical test tube and then injected into gas chromatograph for analysis.

Detection methods for organotin compounds analysis The most common analytical methods used for determining trace organotin compounds are gas chromatography (GC) and liquid chromatography (LC), coupled with various detectors. The trace organotin compounds analyzed are always in a complicated mixture. The selective detector chosen is to avoid interference from the matrix. This section introduces different detectors for GC and LC techniques in organotin compound determination. The common detectors for the GC system used to measure organotin compounds are mass spectrometry (MS) (Cardellicchio et al. 2001; Ikonomou et al. 2002; Muñoz et al. 2004; Colombini et al. 2004; Chou and Lee 2005; Liscio et al. 2009); tandem mass spectrometry (MS/MS) (Tsunoi et al. 2002; Carvalho et al. 2007; Beceiro-González et al. 2009); flame photometric detector (FPD) (Jiang and Liu 2000; Gui-bin and Qun-fang 2000; Bancon-Montigny et al. 2000; Zuliani et al. 2008); atomic absorption spectrometry (AAS) (Bowles et al. 2004; Van et al. 2006); atomic emission detector (AED) (Botana et al. 2002; Campillo et al. 2004; Zachariadis and Rosenberg 2009); and inductively coupled plasma-mass spectrometry (ICPMS) (Tao et al. 1999; Vercauteren et al. 2000, 2001; Üveges et al. 2007). Generally, the derivatization of organotin species is required before analysis by the gas chromatography. Common derivatization reagents used are Grignard reagent, sodium borohydride

273

(NaBH4), sodium tetraethylborate (NaBEt4), and many others. The mass spectrometrybased detectors and flame photometric detector are usually for determination of organometallic compounds; the other detectors are for detection of inorganic compounds. Because of the properties of organometals, the organotin species can be determined by all detectors in the GC system. The detectors, with the exception of the flame photometric detector, have high sensitivity for determining the amount of inorganotin compounds in the chromatogram. In a flame photometric detector, analytes were burned and emitted to the light; the light from the analyte is filtered by a special optical filter. The flame photometric detector is also applied to determine organotin species. The wavelengths of optical filters of 390 nm and 610 nm are used to detect Sn-C bonds and Sn—H bonds (Jiang and Liu 2000; Guibin and Qun-fang 2000; Bancon-Montigny et al. 2000; Zuliani et al. 2008). A filter of 610 nm is commonly used to avoid the interference of sulfur species. The modified detector, pulse flame photometric detector (PFPD) (Bancon-Montigny et al. 2000; Zuliani et al. 2008), is replacing the conventional FPD to detect organotin compounds (Jiang and Liu 2000; Gui-bin and Qun-fang 2000). The PFPD is based on a pulsed flame source and not a continuous one, such as in conventional flame photometric detectors. PFPD tends to have higher selectivity and sensitivity than those of PFD. The detection limits for trace organotin compounds detected by PFPD can be lower than those detected by conventional PFD (Bancon-Montigny et al. 2000). Recently, mass spectrometry was used to determine the organotin compounds because of its high selectivity and sensitivity (Cardellicchio et al. 2001; Ikonomou et al. 2002; Tsunoi et al. 2002; Muñoz et al. 2004; Chou and Lee 2005; Carvalho et al. 2007; Liscio et al. 2009; Beceiro-González et al. 2009). The mass-spectral property of organotin compounds always shows fragment ions

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207 6.0 × 105

[M–C4H9–C2H5]+

Abundance

5.0 × 105 4.0 × 105

263 291

5

177 151

3.0 × 10

2.0 × 105

[M–C2H5]+

289 235

121 119

1.0 × 105 0

100

150

200

250 m/z

300

350

400

Figure 11.2. Mass spectrum of the derivative of tributyltin produced by EI-MS. From J. Chromatogr. A 1064 (2005) 1–8. Used by permission.

as a group of peaks due to the isotopes of tin. The relative isotope abundances of tin are 2.98% for 112Sn, 2.03% for 114Sn, 1.04% for 115 Sn, 44.63% for 116Sn, 23.57% for 117Sn, 74.34% for 118Sn, 26.37% for 119Sn, 100% for 120 Sn, 14.21% for 122Sn, and 17.77% for 124Sn. In mass spectrum, the ion ratio of tin-obtained compound should be matched to the relative isotope abundances of tin. The mass spectra of the organotins are very characteristic because tin has ten isotopes. The mass spectrum of the sodium tetraethyl borate derivative of tributyltin produced by electron impacted ionization (EI) is shown in Figure 11.2. The relative abundances of the isotope cluster ions are obtained. The ion m/z 291 corresponds to an ion of elemental composition 120Sn(C4H9)3. The ion m/z 289 is composed of 118Sn(C4H9)3. For quantitative analysis, the m/z 291 and 289 are selected as the quantitation and confirmation ion, respectively. The novel analytical method of tandem mass spectrometry has been developed for use in trace analysis in recent years. The accuracy of the results can be improved by using selected reaction monitoring (SRM) of tandem mass spectrometry in quantitation of

organotin species (Beceiro-González et al. 2009). The comparison of the detection limits of organotin compounds with various detectors is listed in Table 11.1. The elementspecific detectors including AAS, AED, and ICP-MS are used to detect the amount of metal tin in samples (Tao et al. 1999). The detection of AAS is seldom used currently due to poor sensitivity. The AES detection, especially microwave-induced plasma atomic emission detector (MIP-AED), is commonly used for determination of organotin species (Botana et al. 2002; Campillo et al. 2004; Zachariadis and Rosenberg 2009). The ICP-MS and AED detections give the highest sensitivity in all organotin compounds and inorganotin compounds (Tao et al. 1999). It is also commonly used in the analysis of organotin species. The detection limits with various element-specific detectors for organotin compound analysis are also listed in Table 1.1. Some LC systems with mass spectrometry detectors are proposed for analysis of organotin compounds. The ionization techniques used in the LC-MS system include atmospheric pressure ionization (API) and

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HS-SPME Ultrasonic LLE HS-SDME SPE HS-SPME LLE HS-SPME HS-SPME HS-SPME LLE Ultrasonic LLE Purge & trap — Purge & trap HS-SPME LLE LLE HS-SPME SBSE MAE

Water, soil Marine sediment Water Water Water Sediment Water Lard samples Water Sewage sludge Environmental samples — Water & marine sediments Water Urine Seawater Environmental samples Environmental samples Water & mussel

Sample Preparation

Marine sediment Marine organism Water, sediment, tissue

Sample Matrices

ICP-MS ICP-MS ICP-MS

ICP-MS

AAS AAS AES AES AES & MS

FPD

MS MS MS MS/MS MS/MS MS/MS FPD FPD FPD

MS MS HRMS

Detector

NA 10 fg/L (TPhT) 3.4–6.6 ng Sn /Kg

0.7–1.6 fg

NA 6–7 pg as Sn 2.7–12.5 ng Sn/g NA 0.42–0.67 μg/L

5.1–16.3 ng Sn /g

NA 0.07–0.10 ng Sn/g 0.4–4.6 ng/L 0.26–0.84 ng/L 4–33 ng/L 0.3–1 pg/g 20–200 ng/L NA 0.09–0.48 ng Sn/L

730–969 pg Sn/g 13–25 ng Sn/g 0.13–5.9 pg (MDL)

LOD

Vercauteren et al. 2000 Vercauteren et al. 2001 Üveges et al. 2007

Tao et al. 1999

Bowles et al. 2004 Van et al. 2006 Campillo et al. 2004 Botana et al. 2002 Zachariadis and Rosenberg 2009

Zuliani et al. 2008

Colombini et al. 2004 Muñoz et al. 2004 Chou and Lee 2005 Carvalho et al. 2007 Tsunoi et al. 2002 Beceiro-González et al. 2009 Jiang and Liu 2000 Gui-bin and Qun-fang 2000 Bancon-Montigny et al. 2000

Cardellicchio et al. 2001 Liscio et al. 2009 Ikonomou et al. 2002

References

DcyT, dicyclohexyltin; DMT, dimethyltin; DOT, dioctyltin; DPhT, diphenyltin; MMT, monomethyltin; MOT, monooctyltin; TCT and TcyT, tricyclohexyltin; TeMT, tetramethyltin; TMT, trimethyltin; TOT, trioctyltin; TPhT, triphenyltin; AES, accelerated solvent extraction; SBSE, stir bar sorptive extraction.

MBT, DBT, TBT MBT, DBT, TBT, MBT, DBT, TBT, TeBT, MPhT, DPhT, TPhT, DCyT, TCyT MBT, DBT, TBT MBT, DBT, TBT MBT, DBT, TBT, TeBT, MPhT, TPhT MBT, DBT, TBT, MPhT, DPhT, TPhT MBT, DBT, TBT MBT, DBT, TBT MBT, DBT, TBT, TeBT MMT, DMT, TMT, DOT MBT, DBT, TBT, MPhT, DPhT, TPhT, MOT, DOT, TOT, MBT, DBT, TBT, MPhT, DPhT, TPhT, MOT, DOT, TOT MBT, DBT, TBT MBT, DBT, TBT, MPhT, DPhT, TPhT MBT, DBT, TBT, TeMT, DMT, MPhT MBT, DBT, TBT MBT, DBT, TBT, TeBT, MPhT, DPhT, TPhT MBT, DBT, TBT, TeBT, MPhT, DPhT, TPhT TCT, TPhT TCT, TPhT MBT, DBT, TBT

Species

Table 11.1. Comparison of detection limits of various detectors for organotin compound analysis.

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inductively coupled plasma (ICP) ionization (Wahlen and Catterick 2003; GonzálezToledo et al. 2003; Jones-Lepp and Momplaisir 2005; Wang et al. 2008; Liscio et al. 2009). The advantage of LC-MS analysis is that it is not necessary to perform derivatization for the analytes. Reversed-phase and cation exchange LC columns are often used in trace organotin compound analysis.

Analysis of organotin compounds in various matrices Analysis of organotin compounds in seafood Seafood samples Organotin compounds are highly toxic to many aquatic species and have sensitive endocrine effects. They have been detected in humans who consumed seafood. In 1997, Kannan and Falandysz made a survey of the organotin compound contamination levels in muscle tissue of several fish species in the Baltic Sea. Since 2000, several articles reported various analytical methods to detect the trace organotin compounds in fish, shrimp, and mollusca tissues. The summary of ana-

lyzing organotin compounds in marine biota is shown in Table 11.2. To reduce the uncertainty regarding tributyltin in seafood, Willemsen et al. built an EU-wide database using data on the occurrence of tributyltin in seafood for the European market (Willemsen et al. 2004). Their research determined that, the average tributyltin levels over all species increase in the order of fish, crustaceans, and mollusca. In mollusca, a higher concentration of organotin species was found in bivalves than in cephalopods and gastropods. For crustaceans, only shrimp were analyzed, with low concentrations of organotin compounds detected. The fish of the herring family were the most contaminated. The high levels of organotin species in the mollusca were more correlated with location than with species. The concentrations of organotin compounds detected in mussels and clams from Italy were higher than those obtained from Portugal. The organotin compound concentrations in seafood samples from other countries such as Greece, Spain, France, the Netherlands, and the United Kingdom were very low. For shrimp, a higher average level of organotin compounds was found in Belgian

Table 11.2. Analytical methods for determination of organotin compounds in marine biota. Species

Sample Matrices

Sample Preparation and Derivatization

MBT, DBT, TBT, MPhT, DPhT, TPhT, TeBT TBT, DBT, DPhT, TPhT

Mussel

ASE, NaBEt4

Mussel, oyster Dogfish, lobster Oyster

MBT, DBT, TBT TBT MBT, DBT, TBT, TeBT, DPhT, MPhT, DOT, TPrT, TePrT MBT, DBT, TBT, MPhT, DPhT, TPhT MBT, DBT, TBT, TPrT MBT, DBT, TBT

Detection

LOD

References

GC/MS

6–78 ng Sn/g

SPE

HPLC

3–140 ng Sn/g

SPME, NaBEt4

GC-AES

NA

MAE, NaBEt4

GC/MS

NA

Mussel, oyster

ASE, MAE, NaBEt4

GC/ICPMS LC/ICPMS

NA

Looser et al. 2000 González-Toledo et al. 2001 Tutschku et al. 2002 Monperrus et al. 2003 Wahlen and Catterick 2003

Milkfish

LLE, NaBEt4

GC-FPD

0.2–1.7 ng

Shellfish Mussels

HS-SDME, NaBEt4 LLE, SPE for cleanup, NaBEt4,

GC/ICPMS GC/MS

0.8–1.8 ng/L 13–25 ng/g

Tang and Wang 2007 Xiao et al. 2008 Liscio et al. 2009

DOT, dioctyltin; DPhT, diphenyltin; TeBT, tetrabutyltin; TePrT, tetrapropyltin; TPhT, triphenyltin; TPrT, tripropyltin; ICPMS, inductively coupled plasma mass spectrometry; SDME, single-drop microextraction.

Organotin Compounds Analysis

shrimp compared with those from Spain and Portugal. High contaminations with organotin compounds were found in Greek sardines. The highest concentrations of tributyltin were found in bivalves from Italy and Portugal, sardines from Greece, as well as in shrimp from Belgium. Looking at the generally high organotin compound contamination produced in the Mediterranean near the coast of Italy, the conclusion was that bivalves are unable to metabolize tributyltin. The analytical results for Portuguese bivalves and Belgian shrimp samples indicate lower contamination levels of organotin compounds in the Atlantic Ocean and the North Sea. It was concluded from the study results that sardines may be able to metabolize tributyltin. Seasonal variation in the concentration of butyltin compounds in marine fish That the metabolism of butyltins in fish varies with seasonal changes has been suggested by Beamish (1964). Dong et al. demonstrate that the accumulation of butyltin compounds (BTs) exhibits seasonal variation with respect to the concentrations of organotin compounds and the composition in fish (Dong et al. 2004). Chipps and coworkers suggested that the physiological responses are compatible with environmental conditions, such as the occurrence of reduced food availability and lower activity levels in winter months (Chipps et al. 2000). During certain fast-growing seasons, the test organisms showed an overall decline in pollutant residue concentrations. The metabolic rate of juvenile fish Esox masquinongy in spring and autumn was significantly higher than that during winter months at comparable water temperatures. The low concentration of tributyltin detected in the ponyfish and lizardfish in spring could simply be attributed to dilution resulting from rapid growth. Therefore, it was postulated that the accumulation pattern of butyltins in different seasons and among different organisms could

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be strongly influenced by seasonally mediated physiological changes, such as dilution due to growth and metabolic compensation. Chan et al. (2008) investigated seasonal changes in imposex and tissue burden of butyltin compounds in Thais clavigera populations. The relative penis size index (RPSI) and vas deferens sequence index (VDSI) (which followed the quantification of butyltins in tissues) were used to study imposex in snails. The results showed that concentration of tributyltin in tissue varied with season: the winter samples in general contained much higher concentrations of tributyltin than did the summer samples. The level of butyltin contamination varied with the distance from the Yantian Port, and most of the collected females suffered from imposex. The values of RPSI, VDSI, and organotin concentrations were higher in T. clavigera collected from areas closer to the port. The value of RPSI showed marked seasonal variability, with lower values in the summer season, whereas the values of VDSI exhibited little seasonal variability. Fate of tributyltin during seafood (muscle) preparation It is known that organotin compounds such as tributyltin undergo slow degradation in biota samples when stored for longer periods at ambient temperatures or higher (GomezAriza et al. 1999). Most seafood (except oysters) is consumed after a short-term heating treatment. Long cooking periods are not common because of the resultant rubbery texture, which makes mussels unpleasant to consume. Only the factors of food preparation with a conceivable effect on tributyltin concentration were taken into account. These factors included the following: (1) the manner of heating mussels, by steaming, cooking, microwave, and pan frying and (2) the addition of substances that could have a solvent effect: frying oil and wine because of its alcohol content.

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Mussels were cooked in a microwave, by steaming, and in a frying pan with and without oil or wine (Willemsen et al. 2004). According to each heating method, mussels were cooked for different times and with or without their shells. Mussels expected to have high tributyltin levels were used in order to detect significant changes in concentration. The results from each trial showed that tributyltin never completely degraded after cooking, and in some cases, only a slight decrease of tributyltin concentration was observed. When the mussels were cooked by a microwave oven, steamed, or boiled, the tributyltin concentration was never lower than 70% of the initial concentration. A significant decrease to 40% of the initial concentration was observed, however, after cooking mussels in a frying pan with wine or oil and with their shells. Significant loss seemed to occur only after prolonged heating, a procedure that is not common in most European countries. All in all, there was no significant loss of tributyltin during common mussel home-cooking procedures.

Analysis of organotin compounds in seawater and sediment Organotin compounds are useful as fungicides, bactericides, pesticides, biocides, wood preservatives, and stabilizing agents in various fields as described above. Tributyltin and triphenyltin used as antifouling agents in paints may cause contamination when released into the marine and freshwater environments. Despite the recent bans of organotin compounds, significant concentrations of tributyltin, triphenyltin, and their metabolites can still be found in waters. Puri et al. developed a polymeric adsorbent for efficient preconcentration of organotin compounds from sediment and seawater (Puri et al. 2004). A 100-mg sample of the adsorbent was taken in a glass column fitted with a polyethylene frit. To remove tributyltin chloride completely, the polymeric adsorbent

packed in the column needs to be washed several times with 0.1 M hydrochloride (HCl) in methanol until no tin bleeding is observed. Subsequently, the column is washed with 5 mL methanol to condition the adsorbent before loading the sample. An aliquot of a 10-ng solution of tributyltin chloride, dibutyltin dichloride, monobutylytin trichloride, and triphenyltin chloride compounds was diluted with water to the total volume of 10–15 mL and passed over the adsorbent at the flow rate of 1–3 mL/min. After washing the adsorbent with 2 mL methanol, the retained compounds were eluted with two fractions of 1 mL of methanol containing 0.1 M hydrochloride. The elutant was collected in a tube and measured by graphite furnace atomic absorption spectrometry (GFAAS). Linearity was satisfactory for all organotin compounds measured for a concentration range of absolute Sn of 0.1–4 ng, with a correlation coefficient of more than 0.994. The relative standard derivation was less than 5% for various matrices, with a limit of detection of 30 ng/L. Chou and Lee (2005) presented an in situ ethylation of organotin compounds combined with headspace solid-phase microextraction and gas chromatography-mass spectrometry for analyzing organotin compounds in seawater. The organotin compounds, including monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), tetrabutyltin (TeBT), phenyltin (MPhT), and triphenyltin (TPhT), were in situ derivatized with sodium tetraethylborate and adsorbed on a poly(dimethylsiloxane)coated fused silica fiber. SPME procedures to extract organotin compounds in water were at pH 5, with extraction and derivatization simultaneously at 45°C for 30 minutes in a 2% sodium tetraethylborate solution (volume derivatizing agent/volume sample 1 : 1). The linearity ranged from 10 to 10,000 ng/L, with a relative standard deviation less than 12%. The limits of detection of organotin compounds studied were lower than nanograms per liter. This method was used to analyze the amounts of organotin compounds in seawater

Organotin Compounds Analysis

279

Figure 11.3. Mass ion chromatograms of seawater samples taken from Taichung muddy shore (a) Su-Ao harbor, (b) Taichung harbor, (c) produced by HS-SPME-GC-MS.

from the harbors of Taiwan. Figure 11.3 shows the mass ion chromatograms of these seawater samples, and Table 11.3 shows the analytical results. Devos et al. (2005) described an automated analytical method to simultaneously determine six organotin species (MBT, DBT, TBT, MPhT, DPhT, and TPhT) in water and sediment using derivatization with sodium tetraethylborate (NaBEt4) followed by automated HS-SPME combined with GC-MS. Home-synthesized deuterated organotin compound analogs were used as internal standard. Five milliliter seawater, 5 mL buffer solution, and 1 mL methanol were

mixed in a closed-cap headspace vial of 20 mL. Subsequently, 250 ng/L of six kinds of internal standards were added and mixed with sample solution. Derivatization was performed by adding 1% NaBEt4 solution into sample vials. The sample vials were vigorously shaken and placed in the ultrasonic bath for 10 min. Finally, the vials were placed in the MPS-2 autosampler for HS-SPME extraction and analyzed by GC-MS. The limits of quantification (LOQs) ranged from 1.3 to 15 ng/L for water samples and from 1.0 to 6.3 μg/kg for sediment samples. Campillo and his colleagues described a procedure for the simultaneous determination

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Table 11.3. Concentrations of organotin species in surface seawater (ng/L). Real Water Sample

Monobutyltin

Dibutyltin

Tributyltin

Tetrabutyltin

Monophenyltin

Triphenyltin

Taichung harbor Taichung muddy shore Kaohsiung harbor Kaohsiung sand beach Keelung wharf Su-Ao harbor

ND 54 ± 3

ND 102 ± 12

ND 48 ± 6

ND 277 ± 30

ND 298 ± 28

ND 16 ± 4

ND ND

83 ± 9 25 ± 3

ND ND

216 ± 23 107 ± 11

186 ± 17 86 ± 9

ND ND

65 ± 6 99 ± 8

160 ± 19 24 ± 2

ND 148 ± 16

287 ± 27 34 ± 4

ND 64 ± 7

ND ND

Note: The samples were collected at harbors around the Taiwanese coast and filtered with 0.2 μm nylon 66-membrane filter. Every sample had triplicate measurement. ND, not detected.

of six organotin compounds, including methyltin, butyltin, and phenyltin in water and marine sediments by purge and trap extraction followed by capillary gas chromatography with atomic emission detection (Campillo et al. 2004). For a water sample, 10 mL of sample mixed with 10 μL of methanol was placed in a 50 mL polycarbonate reaction flask. The pH was adjusted to 4.8 using 1 mL of 1.5 M acetate buffer solution. One hundred microliters of 0.2% (w/v) NaBEt4 derivatization solution was added. The solution was agitated for 10 minutes at 800 rpm on a rotary shaker. For sediment samples, 2 grams of sediment were accurately weighed and placed in a 50-mL centrifugation tube. Ten milliliters of a mixture of concentrated acetic acid/methanol (1 : 9) was added and then sonicated for 30 s. The mixture was centrifuged at 3000 rpm for 5 min. One milliliter of the supernatant solution was mixed with 50 mL of a solution containing 5 mL of acetate buffer solution and 500 μL of 0.2% (w/v) NaBEt4 solution. This reaction was carried out similarly with the water sample. Finally, 5 mL of the mixture was submitted to the purge and trap process. The linearity ranged from 0.05 to 3.0 ng/L for DMT, MBT, DBT, MPhT and TBT, and from 0.2 to 10 ng/L for TeMT. Detection limits ranged from 11 to 50 ng/L in the water sample and from 2.7 to 12.5 ng/g in sediment for tributyltin and tetramethyltin.

Ikonomou et al. (2002) presented a gas chromatography method combined with high-resolution mass spectrometry (GCHRMS) for the accurate and sensitive determination of nine organotin species in sediment and water samples: tetrabutyltin, tributyltin, dibutyltin, monobutyltin, triphenyltin, diphenyltin, monophenyltin, tricyclohexyltin, and dicyclohexyltin. For water sample pretreatment, 100 mL of the water sample was mixed with 10 mL of pH 4.5 acetate buffer solution and 100 ng Di-(npropyl)tin (DPrT) as internal standard (I.S.). Then, 1 mL mixture was derivatized with a 0.5-mL sodium tetraethylborate solution (1%, w/v). After ethylation, the derivatives of the organotin compounds were extracted with 2 × 50 mL hexane and then reduced to 0.3 mL by nitrogen gas. For the sediment sample, 1 to 2 g of sediment were weighed and mixed with I.S., 5 mL of 30% NaCl solution, 2 mL of sodium acetate buffer (pH 4.5), and appropriate glacial acetic acid in a 60-mL vial. The acidic mixture was extracted with diethylether (Et2O)/hexane (8 : 2) by shaking for 1 hour. After centrifugation, 1 mL of supernatant was taken to mix with 0.5 mL of 1% (w/v) sodium tetraethylborate for ethylation. The derivatives of organotin were transferred and extracted with Et2O/hexane (2 : 8) and then analyzed by GC-HRMS. The limits of detection were 7–29 pg/g for water and 0.35–1.45 pg/g for sediments.

Organotin Compounds Analysis

During sediment analysis, some matrix effects were observed. High levels of sulfur and its organic derivatives in some cases can affect detection and quantification of organotin compounds by formation of volatile compounds. Bravo and his colleagues (2004) proposed the reoptimization of GC parameters and application of solid-phase microextraction (SPME) to resolve the analytical problems of sulfur interference. Furthermore, Wasik and coworkers (2007) developed a simple method for elimination of sulfur matrices using pressurized liquid extraction.

Analysis of organotin compounds in wastewater and sewage sludge Organotin compounds may lead to contamination of municipal wastewater and sewage sludge because these compounds are found in various applications of industry and agriculture. Zuliani et al. (2008) studied the conditions of pulsed-flame photometric detection for the determination of organotin compounds in sewage sludge. The results show good agreement between determined and certified values. Sewage sludge from a local wastewater treatment plant was analyzed by this method. The limits of detection for organotin compounds including butyltins, phenyltins, and octyltins ranged from 5 to 16 ng/g. Vreysen et al. (2008) investigated the removal of dibutyltin and tributyltin from shipyard wastewaters by a one-step adsorption–flocculation method. The results showed that the water treatment process that removes both dissolved pollutants and paint flakes is evaluated on technical, ecological, and economic criteria. Hence, an adsorption– flocculation process using a mixture of two adsorbents (a clay-based adsorbent and a powdered activated carbon) was optimized for the best adsorbent and pollutant removal. The optimal conditions were evaluated with both artificial and real shipyard wastewater. The cost of the adsorption–flocculation process in relation to different influent con-

281

centrations and discharge limits was also estimated. Díez et al. (2006) indicated that the sediments were assessed by the quantitative determination of butyltins as markers of urban and industrial wastewater contamination. The limit of detection was in the range of 1.3–11.8 ng/g. Arnold et al. (1998) studied two new methods for the simultaneous identification and quantification of organotin compounds, including monobutyltin, dibutyltin, tributyltin, monophenyltin, diphenyltin, triphenyltin, and tricyclohexyltin in natural waters and sediments. The method used aqueous ethylation followed by liquid–liquid extraction or solid-phase extraction and large volumeinjection GC-MS of water samples. The results showed excellent precision (relative standard deviations <5%) and low method detection limits ranging from 0.3 to 1.5 ng/L for 50 mL of aqueous samples and from 0.4 to 2 ng/g for 2.5 g of dried sediments. CarlierPinasseau et al. (1997) developed a reliable and rapid speciation method for the determination of both butyltin and phenyltin compounds in environmental samples. The analytical procedure is based on a one-step simultaneous ethylation/extraction using the reaction with sodium tetraethylborate in an aqueous phase in the presence of iso-octane. Then, a direct analysis of the organic layer is performed using gas chromatography interfaced to flame photometric detection (Díez et al. 2006; Carlier-Pinasseau et al. 1997). Detection limits of a few picograms of tin are reached. The technique has been validated by its application to different polluted environmental samples: wastewaters, sediments, and biological tissues.

Analysis of organotin compounds in other matrices Analysis of organotin compounds in human body fluids The choice of human fluid is critical for evaluating the toxicity of ingesting contaminated

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food. Urine is a commonly selected biological matrix for drug metabolism because it is a relatively easy, noninvasive collection method. Development of analytical methods of organotin compounds in urine is a route to establishing a pathway of organotin compounds in the human body. Nevertheless, only a few articles discuss organotin compounds in urine. Zachariadis and Rosenberg (2009) provided a simple pretreatment for butyltins and phenyltins in urine. Liquid–liquid extraction and headspace solid-phase microextraction were used to extract organotin compounds in urine. In liquid–liquid extraction, 2.5 mL of urine sample was added 0.25 mL of hexane and 25 μL of sodium tetraethylborate, then manually agitated for 3 minutes at room temperature. Detection was gas chromatography coupled with a microwave-induced plasma atomic emission detector (GC-MIP-AED) analysis. Relative standard deviations were between 4.2% and 11.7% in 5.0 μg/mL. In headspace solid-phase microextraction, optimized conditions were 25 mL of urine with 25 μL of sodium tetraethylborate in a 40-mL vial. Subsequently, PDMS-coated fused silica fiber was exposed to the headspace of the solution for 15 min, followed by GC-MIPAED analysis. Relative standard deviations of this method were between 3.7% and 11.4%. In these results, LLE-GC-MIP-AED provided a quick way of screening organotin compounds in undiluted human urine. HS-SPME-GC-MIP-AED was an in situ derivatization and extraction method proposed for undiluted human samples in the low nanograms per liter range. Mino et al. (2008) described a procedure to investigate the toxicity of contaminated human breast milk to newborn babies. Four milliliters of breast milk sample was extracted with 25 mL hexane/diethyl ether (4 : 6) for 30 minutes. The extract was then evaporated by a rotary evaporator under 25°C to near dryness. The sample residue was then dissolved in 10 mL of ethanol. The ethanol solu-

tion subsequently was passed through a cation exchange SPE cartridge (Amberlite CG-120 Type 1) as a cleanup procedure. The eluent was derivatized with Grignard reagent. Analysis with gas chromatography-flame photometric detector followed. The limits of detection of the proposed method were 2.5 ng/ mL for monobutyltin and 1.3 ng/mL for other organotin compounds. This is the first study to demonstrate organotin pollution of human breast milk. Analysis of organotin compounds in soils Organotin-based pesticides causing pollution were widely used. Soil is one of environmental matrices that has attracted much attention due to its adsorption of organic matter and minerals in the matrix. Zuliani et al. (2006) developed a HS-SPME method to analyze organotin compounds in soils. Soil samples of 0.5 to 1 g were pre-extracted by 20 mL of glacial acetic acid for 16 hours. The extract was added to 0.2 mL of 2% sodium tetraethylborate and a 60-mL sodium acetate buffer and shaken for 10 min. The PDMS-coated fiber was introduced for 30 minutes and was followed by gas chromatography-pulsed flame photometric detector (GC-PFPD) analysis. According to different physicochemical properties of each soil and severe matrix interference, a standard additional method was applied to improve the method’s performance. The recoveries of the proposed method exceeded 80%, and the limits of detection and limits of quantification were both in the nanograms of Sn per gram level. Heroult et al. (2008) investigated various extraction protocols, such as mechanical stirring (MSAE), ultrasonic extraction (UAE), accelerated solvent extraction (ASE), and microwave-assisted extraction (MAE) for solid samples analysis. In MSAE, 1 g of sample was added to 10 mL of ethanoic acid and stirred at 400 rpm for 15 hours. When UAE was used, the mixture was sonicated at 40 kHz for 5 or 30 min. In ASE, 1 g of sample

Organotin Compounds Analysis

was added to 10 g of quartz sand and filled with 20 mL of MeOH/acetic acid (85 : 15). Extraction was carried out three times at 100°C and 90 bars. In MAE, 1 g of sample was added to 10 mL of ethanoic acid and extracted at 40 W for 2 min. After the extraction steps, 1 mL of extract was added to 0.2– 1 mL NaBEt4, 20 mL of sodium acetate buffer, and 1 mL of iso-octane. The mixture was shaken at 400 rpm for 30 minutes. Heroult et al. (2008) indicated that MSAE represents a better recovery (93%–115%) than other protocols (12%–460%). They suggested that MSAE-GC-PFPD is a reliable and accurate procedure, suitable for routine organotin compound analysis in soils. Analysis of organotin compounds in textiles and plastics Organotin compounds have also been extensively used in cotton fabric moth-proofing and in UV oxidation resistance of textiles. Niu et al. (2006) described an ultrasonic extraction for the analysis of nine organotin compounds in textiles. Two grams of sample was pre-extracted with 40 mL of 0.2% sodium diethyldithiocarbamate (NaDDTC) aqueous solution. Extract (1 mL) was added to 5 mL of sodium acetate buffer and 2 mL of 2% sodium tetraethylborate and then sonicated for 15 min. The solution was then added to 2 mL of hexane for ultrasonic extraction, followed by gas chromatography-mass spectrometry analysis. Relative standard deviations of this method were between 2.9% and 7.3% at 0.25 mg/kg. In their study, orthogonal array design was used to investigate the optimized derivatization conditions, including reaction time, sodium tetraethylborate amount, and pH value. Wang et al. (2008) developed a microwaveassisted extraction combined with liquid chromatography-mass spectrometry (MAELC-MS) method for analysis of organotin compounds in textiles and plastics. A sample was added to 30 mL of a 60% methanol solu-

283

tion and irradiated at 90°C for 5 min. The extract was concentrated to about 2 mL by rotary evaporator at 40°C and diluted to 8 mL with mobile phase, followed by liquid chromatography-electrospray ionizationmass spectrometry (LC-ESI-MS) analysis. Relative standard deviations of the method were lower than 9%. LC-MS analysis prevented the derivatization with hazard materials. MAE-LC-MS is a rapid and accurate procedure to analyze organotin compounds. The recoveries of organotin compounds in textile and plastic samples were in the range of 55%–95%, and the relative standard derivation was 3%–9%. Li et al. (2009) established an ultrasonic extraction of organotin analytes in PVC plastics. A sample (0.5 g) was dissolved in 5 mL of tetrahydrofuran and underwent ultrasonic extraction for 15 minutes. Next, 10 mL of methanol was added. After centrifugation, 1 mL of supernatant was sonicated with 2 mL of 2% sodium tetraethylborate and 5 mL of sodium acetate buffer for 15 minutes. After derivatization, 2 mL of hexane was added to extract derivatives by ultrasonic extraction for 10 minutes, followed by gas chromatographymass spectrometry analysis. This method was a simple and rapid procedure for analyzing ten organotin compounds in PVC plastics.

Conclusion Organotin compounds are widely used in industry and agriculture. They are found to injure organisms and affect marine life, as well as birds, mammals, and humans through the food chain. Organotin compounds are considered a class of the endocrine-disrupting chemicals because they cause imposex effects. Many analytical methods were developed to analyze trace organotin compounds in various matrices, including seafood, water, sediment, human body fluid, and soil, in order to study the effects of organotin compounds on human health and environmental safety. Sample preparation and detection are the

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most important steps in an analytical method. In this chapter, some samples of techniques to extract trace organotin compounds were introduced, such as liquid–liquid extraction, solid-phase extraction, solid-phase microextraction, and liquid-phase microextraction. Also, different detection techniques, including GC-MS, GC-ICP-MS, LC-MS, etc., were described. The results allow evaluation of the distribution of trace organotin compounds in the environment, food, and humans, and biological effects on organisms.

References Aguerre, S.; Lespes, G.; Potin-Gautier, M. (2003) Physico-chemical approach to study organotin sorption-desorption during solid-phase microextraction. J Chromatogr A. 999, 61–70. Arnold, C.G.; Berg, M.; Müller, S.R.; Dommann, U.; Schwarzenbach, R.P. (1998) Determination of organotin compounds in water, sediments, and sewage sludge using perdeuterated internal standards, accelerated solvent extraction, and large-volume-injection GC/ MS. Anal Chem. 70, 3094–3101. Bancon-Montigny, C.; Lespes, G.; Potin-Gautier, M. (2000) Improved routine speciation of organotin compounds in environmental samples by pulsed flame photometric detection. J Chromatogr A. 896, 149–158. Beamish, F.W.H. (1964) Seasonal changes in standard rate of oxygen consumption of fishes. Can J Zool. 42, 189–194. Beceiro-González, E.; Guimaraes, A.; Alpendurada, M.F. (2009) Optimisation of a headspace-solid-phase micro-extraction method for simultaneous determination of organometallic compounds of mercury, lead, and tin in water by gas chromatography-tandem mass spectrometry. J Chromatogr A. 1216, 5563–5569. Belfroid, A.C.; Purperhart, M.; Ariese, F. (2000) Organotin levels in seafood. Mar Pollut Bull. 40, 226–232. Birjandi, A.P.; Bidari, A.; Rezaei, F.; Hosseini, M.R.M.; Assadi, Y. (2008) Speciation of butyl and phenyltin compounds using dispersive liquid-liquid microextraction and gas chromatography-flame photometric detection. J Chromatogr A. 1193, 19–25. Botana, J.C.; Rodríguez, R.R.; Díaz, A.M.C.; Ferreira, R.A.L.; Torrijos, R.C.; Pereiro, I.R. (2002) Fast and simultaneous determination of tin and mercury species using SPME, multicapillary gas chromatography and MIP-AES detection. J Anal At Spectrom. 17, 904–907. Bowles, K.C.; Tiltman, M.D.; Apte, S.C.; Hales, L.T.; Kalman, J. (2004) Determination of butyltins in environmental samples using sodium tetraethylborate derivatisation: Characterization and minimization of interferences. Anal Chim Acta. 509, 127–135.

Bravo, M.; Lespes, G.; Gregori, I.D.; Pinochet, H.; PotinGautier, M. (2004) Identification of sulfur interferences during organotin determination in harbour sediment samples by sodium tetraethyl borate ethylation and gas chromatography-pulsed flame photometric detection. J Chromatogr A. 1046, 217–224. Campillo, N.; Aguinaga, N.; Viñas, P.; López-García, I.; Hernández-Córdoba, M. (2004) Speciation of organotin compounds in waters and marine sediments using purge-and-trap capillary gas chromatography with atomic emission detection. Anal Chim Acta. 525, 273–280. Cardellicchio, N.; Giandomenico, S.; Decataldo, A.; Leo, A.D. (2001) Speciation of butyltin compounds in marine sediments with headspace solid phase microextraction and gas chromatography-mass spectrometry. Fresenius J Anal Chem. 369, 510–515. Carlier-Pinasseau, C.; Lespes, G.; Astruc, M. (1997) Validation of organotin compound determination in environmental samples using NaBEt4 ethylation and GC-FPD. Environ Technol. 18, 1179–1186. Carvalho, P.N., Pinto, L.F., Basto, M.C.P., Vasconcelos, M.T.S.D. (2007) Headspace solid-phase microextraction and gas chromatography-ion trap tandem mass spectrometry method for butyltin analysis in sediments: Optimization and validation. Microchem J. 87, 147–153. Chan, K.M.; Leung, K.M.Y.; Cheung, K.C.; Wong, M.H.; Qiu, J.-W. (2008) Seasonal changes in imposex and tissue burden of butyltin compounds in Thais clavigera populations along the coastal area of Mirs Bay, China. Mar Pollut Bull. 57, 645–651. Chipps, S.R.; Clapp, D.F.; Wahl, D.H. (2000) Variation in routine metabolism of juvenile muskellunge: Evidence for seasonal metabolic compensation in fishes. J Fish Biol. 56, 311–318. Chou, C.C.; Lee, M.R. (2005) Determination of organotin compounds in water by headspace solid phase microextraction with gas chromatography-mass spectrometry. J Chromatogr A. 1064, 1–8. Colombini, V.; Bancon-Montigny, C.; Yang, L.; Maxwell, P.; Sturgeon, R.E.; Mester, Z. (2004) Headspace single-drop microextraction for the detection of organotin compounds. Talanta. 63, 555–560. Cukrowska, E.; Chimuka, L.; Nsengimana, H.; Kwaramba, V. (2004) Application of supported liquid membrane probe for extraction and preconcentration of organotin compounds from environmental water samples. Anal Chim Acta. 523, 141–147. de Mora, S.J.; Fowler, S.W.; Cassi, R.; Tolosa, I. (2003) Assessment of organotin contamination in marine sediments and biota from the Gulf and adjacent region. Mar Pollut Bull. 46, 401–409. Devos, C.; Vliegen, M.; Willaert, B.; David, F.; Mones, L.; Sandra, P. (2005) Automated headspace-solidphase micro extraction-retention time locked-isotope dilution gas chromatography-mass spectrometry for the analysis of organotin compounds in water and sediment samples. J Chromatogr A. 1079, 408–414. Díez, S.; Lacorte, S.; Viana, P.; Barceló, D.; Bayona, J.M. (2005) Survey of organotin compounds in rivers and coastal environments in Portugal 1999–2000. Environ Pollut. 136, 525–536.

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Díez, S.; Jover, E.; Albaigés, J.; Bayona, J.M. (2006) Occurrence and degradation of butyltins and wastewater marker compounds in sediments from Barcelona Harbor, Spain. Environ Int. 32, 858–865. Donard, O.F.X.; Lalere, B.; Martin, F.; Łobin´ ski, R. (1995) Microwave-assisted leaching of organotin compounds from sediments for speciation analysis. Anal Chem. 67, 4250–4254. Dong, C.D.; Chen, C.W.; Liu, L.L. (2004) Seasonal variation in the composition and concentration of butyltin compounds in marine fish of Taiwan. Environ Pollut. 131, 509–514. Dorneles, P.R.; Lailson-Brito, J.; Fernandez, M.A.S.; Vidal, L.G.; Barbosa, L.A.; Azevedo, A.F.; Fragoso, A.B.L.; Torres, J.P.M.; Malm, O. (2008) Evaluation of cetacean exposure to organotin compounds in Brazilian waters through hepatic total tin concentrations. Environ Pollut. 156, 1268–1276. Eiden, R.; Schöler, H.F.; Gastner, M. (1998) In situ ethylation-purge and programmed-temperaturevaporizer cold trapping-gas chromatography-mass spectrometry as an automated technique for the determination of methyl- and butyltin compounds in aqueous samples. J Chromatogr A. 809, 151–157. Encinar, J.R.; Villar, M.I.M.; Santamaría, V.G.; Alonso, J.I.G.; Sanz-Medel, A. (2001) Simultaneous determination of mono-, di-, and tributyltin in sediments by isotope dilution analysis using gas chromatographyICPMS. Anal Chem. 73, 3174–3180. Encinar, J.R.; Rodríguez-González, P.; Fernández, J.R.; Alonso, J.I.G.; Díez, S.; Bayona, J.M.; Sanz-Medel, A. (2002) Evaluation of accelerated solvent extraction for butyltin speciation in PACS-2 CRM using doublespike isotope dilution-GC/ICPMS. Anal Chem. 74, 5237–5242. Evans, O.; Kauffman, P.; Vonderheide, A.P.; Wymer, L.J.; Morgan, J.N. (2009) The determination of pesticidal and non-pesticidal organotin compounds in water matrices by in situ ethylation and gas chromatography with pulsed flame photometric detection. Microchem J. 92, 155–164. Formme, H.; Mattulat, A.; Lahrz, T.; Ruden, H. (2005) Occurrence of organotin compounds in house dust in Berlin, Germany. Chemosphere. 58, 1377–1383. Gomez-Ariza, J.L.; Giráldez, I.; Morales, E.; Ariese, F.; Cofino, W.; Quevauviller, P. (1999) Stability and storage problems in organotin speciation in environmental samples. J Environ Monit. 1, 197–202. González-Toledo, E.; Benzi, M.; Compañó, R.; Granados, M.; Prat, M.D. (2001) Speciation of organotin compounds in shellfish by liquid chromatographyfluorimetric detection. Anal Chim Acta. 443, 183–190. González-Toledo, E.; Compañó, R.; Prat, M.D.; Granados, M. (2002) Determination of triorganotin species in water samples by liquid chromatographymass spectrometry. J Chromatogr A. 946, 1–8. González-Toledo, E.; Compañó, R.; Granados, M.; Prat, M.D. (2003) Detection techniques in speciation analysis of organotin compounds by liquid chromatography. TrAC-Trends Anal Chem. 22, 26–33. Gui-bin, J.; Qun-fang, Z. (2000) Direct Grignard pentylation of organotin-contaminated lard samples fol-

285

lowed by capillary gas chromatography with flame photometric detection. J Chromatogr A. 886, 197–205. Heroult, J.; Bueno, M.; Potin-Gautier, M.; Lespes, G. (2008) Organotin speciation in French brandies and wines by solid-phase microextraction and gas chromatography-pulsed flame photometric detection. J Chromatogr A. 1180, 122–134. Heroult, J.; Zuliani, T.; Bueno, M.; Denaix , L.; Lespes, G. (2008) Analytical advances in butyl-, phenyl- and octyltin speciation analysis in soil by GC-PFPD. Talanta. 75, 486–493. Hoch, M. (2001) Organotin compounds in the environment–an overview. Appl Geochem. 16, 719–743. Huang, J.-H.; Schwesig, D.; Matzner, E. (2004) Organotin compounds in precipitation, fog, and soils of a forested ecosystem in Germany. Environ Pollut. 130, 177–186. Ikonomou, M.G.; Fernandez, M.P.; He, T.; Cullon, D. (2002) Gas chromatography-high-resolution mass spectrometry based method for the simultaneous determination of nine organotin compounds in water, sediment, and tissue. J Chromatogr A. 975, 319–333. Jiang, G.B.; Liu, J.Y. (2000) Determination of butyltin compounds in aqueous samples by gas chromatography with flame photometric detector and headspace solid-phase microextraction after in-situ hydride derivatization. Anal Sci. 16, 585–588. Jiang, G.B.; Liu, J.Y.; Yang, K.W. (2000) Speciation analysis of titanium dioxide solid powder by fluorination assisted electrothermal vaporization inductively coupled plasma atomic emission spectrometry. Anal Chim Acta. 421, 67–74. Jones-Lepp, T.L.; Momplaisir, G.-M. (2005) New applications of LC-MS and LC-MS2 toward understanding the environmental fate of organometallics. TrACTrends Anal Chem. 24, 590–595. Kannan, K.; Falandysz, J. (1997) Butyltin residues in sediment, fish, fish-eating birds, harbour porpoise, and human tissues from the Polish coast of the Baltic Sea. Mar Pollut Bull. 34, 203–207. Lee, C.-C.; Wang, T.; Hsieh, C.-Y.; Tien, C.-J. (2005) Organotin contamination in fishes with different living patterns and its implications for human health risk in Taiwan. Environ Polluti. 137, 198–208. Lespes, G.; Desauziers, V.; Montigny, C.; Potin-Gautier, M. (1998) Optimization of solid-phase microextraction for the speciation of butyl- and phenyltins using experimental designs. J Chromatogr A. 826, 67–76. Li, Y.; Li, B.; Liu, L.; Zhang, C.; Wu, J.W.; Liu, Z.H.; Li, X.T. (2009) Simultaneous determination of ten organotin compounds in polyvinyl chloride plastics using gas chromatography-mass spectrometry. Chinese J Chromatogr. 27, 69–73. Liscio, C.; Di Carro, M.; Magi, E. (2009) Comparison of two analytical methods for the determination of organotin compounds in marine organisms. C R Chimie. 12, 831–840. Liu, J.Y.; Jiang, G.B. (2002) Survey on the presence of butyltin compounds in Chinese alcoholic beverages, determined by using headspace solid-phase microextraction coupled with gas chromatography−flame

286

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photometric detection. J Agric Food Chem. 50, 6683–6687. Looser, P.W.; Berg, M.; Fent, K.; Mu1hlemann, J.; Schwarzenbach, R.P. (2000) Phenyl- and butyltin analysis in small biological samples by cold methanolic digestion and GC/MS. Anal Chem. 72, 5136– 5141. Meng, P.J.; Lin, J.; Liu, L.-L. (2009) Aquatic organotin pollution in Taiwan. J Environ Manage. 90, S8–S15. Millán, E.; Pawliszyn, J. (2000) Determination of butyltin species in water and sediment by solid-phase microextraction-gas chromatography-flame ionization detection. J Chromatogr A. 873, 63–71. Mino, Y.; Amano, F.; Yoshioka, T.; Konishi, Y. (2008) Determination of organotins in human breast milk by gas chromatography with flame photometric detection. J Health Sci. 54, 224–228. Mizuishi, K.; Takeuchi, M.; Hobo, T. (1998) Trace analysis of tributyltin and triphenyltin compounds in sea water by gas chromatography-negative ion chemical ionization mass spectrometry. J Chromatogr A. 800, 267–273. Moens, L.; Smaele, T.D.; Dams, R.; Broeck, P.V.D.; Sandra, P. (1997) Sensitive, simultaneous determination of organomercury, -lead, and -tin compounds with headspace solid phase microextraction capillary gas chromatography combined with inductively coupled plasma mass spectrometry. Anal Chem. 69, 1604– 1611. Monperrus, M.; Martin-Doimeadios, R.C.R.; Scancar, J.; Amouroux, D.; Donard, O.F.X. (2003) Simultaneous sample preparation and species-specific isotope dilution mass spectrometry analysis of monomethylmercury and tributyltin in a certified oyster tissue. Anal Chem. 75, 4095–4102. Montigny, C.; Lespes, G.; Potin-Gautier, M. (1998) Matrix effects and selectivity of the detector in the determination of butyl- and phenyltins by gas chromatography-flame photometric detection. J Chromatogr A. 819, 221–230. Muñoz, J.; Baena, J.R.; Gallego, M.; Valcárcel, M. (2004) Speciation of butyltin compounds in marine sediments by preconcentration on C60 and gas chromatography-mass spectrometry. J Chromatogr A. 1023, 175–181. Muñoz, J.; Gallego, M.; Valcárcel, M. (2005) Speciation of organometallic compounds in environmetal samples by gas chromatography after flow preconcentration on fullerenes and nanotubes. Anal Chem. 77, 5389– 5395. Nikolaou, A.D.; Gatidou, G.M.; Golfinopoulos, S.K.; Thomaidis, N.; Lekkas, T.D. (2007) A one-year survey of organotin compounds in the reservoirs supplying the drinking water treatment plants of Athens, Greece. Desalination. 210, 24–30. Niu, Z.-Y.; Yuan L.-L.; Ye, X.-W.; Li, L.-S. (2006) Determination of organotin compounds in textiles by GC-MS. J Textiles Res. 27, 22–27. Pereiro, I.R.; Schmitt, V.O.; Szpunar, J.; Donard, O.F.X.; Łobin´ ski, R. (1996) Speciation analysis for organotin compounds in biomaterials after integrated dissolution, extraction, and derivatization in a focused microwave field. Anal Chem. 68, 4135–4140.

Puri, B.K.; Munoz-Olivas, R.; Camara, C. (2004) A new polymeric adsorbent for screening and preconcentration of organotin compounds in sediments and seawater samples. Spectrochimica Acta Part B. 59, 209–214. Rodriguez, I.; Mounicou, S.; Łobin´ ski, R.; Sidelnikov, V.; Patrushev, Y.; Yamanaka, M. (1999) Speciesselective analysis by microcolumn multicapillary gas chromatography with inductively coupled plasma mass spectrometric detection. Anal Chem. 71, 4534– 4543. Shim, W.J.; Yim, U.H.; Kim, N.S.; Hong, S.H.; Oh, J.R.; Jeon, J.K.; Okamura, H. (2005) Accumulation of butyl- and phenyltin compounds in starfish and bivalves from the coastal environment of Korea. Environ Pollut. 133, 489–499. Shioji, H.; Tsunoi, S.; Harino, H.; Tanaka, M. (2004) Liquid-phase microextraction of tributyltin and triphenyltin coupled with gas chromatography-tandem mass spectrometry: comparison between 4-fluorophenyl and ethyl derivatizations. J Chromatogr A. 1048, 81–88. Soderquist, C.J.; Crosby, D.G. (1980) Degradation of triphenyltin hydroxide in water. J Agric Food Chem. 28, 111–117. Stephenson, M.D.; Smith, D.R. (1988) Determination of tributyltin in tissues and sediments by graphite furnace atomic absorption spectrometry. Anal Chem. 60, 696–698. Sullivan, J.J.; Torkelson, J.D.; Wekell, M.M.; Hollingworth, T.A.; Saxton, W.L.; Miller, G.A. (1988) Determination of tri-n-butyltin and di-n-butyltin in fish as hydride derivatives by reaction gas chromatography. Anal Chem. 60, 626–630. Szpunar, J.; Ceulemans, M.; Schmitt, V.O.; Adams, F.C.; Łobin´ ski, R. (1996) Microwave-accelerated speciation analysis for butyltin compounds in sediments and biomaterials by large volume injection capillary gas chromatography quartz furnace atomic absorption spectrometry. Anal Chim Acta. 332, 225–232. Tang, C.H.; Wang, W.H. (2007) Optimization of an analytical method for determining organotin compounds in fish tissue by base-hydrolysis pretreatment and simultaneous ethylation-extraction procedures. Anal Chim Acta. 581, 370–376. Tao, H.; Rajendran, R.B.; Quetel, C.R.; Nakazato, T.; Tominaga, M.; Miyazaki, A. (1999) Tin speciation in the femtogram range in open ocean seawater by gas chromatography/inductively coupled plasma mass spectrometry using a shield torch at normal plasma conditions. Anal Chem. 71, 4208–4215. Tsunoi, S.; Matoba, T.; Shioji, H.; Huong Giang, L.T.; Harino, H.; Tanaka, M. (2002) Analysis of organotin compounds by Grignard derivatization and gas chromatography-ion trap tandem mass spectrometry. J Chromatogr A. 962, 197–206. Tutschku, S.; Schantz, M.M.; Wise, S.A. (2002) Determination of methylmercury and butyltin compounds in marine biota and sediments using microwave-assisted acid extraction, solid-phase microextraction, and gas chromatography with microwave-induced plasma atomic emission spectrometric detection. Anal Chem. 74, 4694–4701.

Organotin Compounds Analysis

Üveges, M.; Abrankó, L.; Fodor, P. (2007) Optimization of GC-ICPMS system parameters for the determination of butyltin compounds in Hungarian freshwater origin sediment and mussel samples. Talanta. 73, 490–467. Van, D.N.; Radziuk, B.; Frech, W. (2006) A comparison between continuum- and line source AAS for speciation analysis of butyl- and phenyltin compounds. J Anal At Spectrom. 21, 708–711. Vercauteren, J.; Meester, A.D.; Smaele, T.D.; Vanhaecke, F.; Moens, L.; Dams, R.; Sandra, P. (2000) Headspace solid-phase microextraction-capillary gas chromatography-ICP mass spectrometry for the determination of the organotin pesticide fentin in environmental samples. J Anal At Spectrom. 15, 651–656. Vercauteren, J.; Peres, C.; Devos, C.; Sandra, P.; Vanhaecke, F.; Moens, L. (2001) Stir bar sorptive extraction for the determination of ppq-level traces of organotin compounds in environmental samples with thermal desorption-capillary gas chromatography−ICP mass spectrometry. Anal Chem. 73, 1509–1514. Vreysen, S.; Maes, A.; Wullaert, H. (2008) Removal of organotin compounds, Cu and Zn from shipyard wastewaters by adsorption-flocclation: A technical and economical analysis. Mar Pollut Bull. 56, 106– 115. Wahlen, R.; Catterick, T. (2003) Sensitive liquid chromatography-mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma. J Chromatogr B. 783, 221–229. Wang, X.; Jin, H.; Ding, L.; Zhang, H.; Zhang, H.; Qu, C.; Yu, A. (2008) Organotin speciation in textile and plastics by microwave-assisted extraction HPLC-ESIMS. Talanta. 75, 556–563.

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Wasik, A.; Radke, B.; Bolalek, J.; Namie´s nik, J. (2007) Optimisation of pressurised liquid extraction for elimination of sulphur interferences during determination of organotin compounds in sulphur-rich sediments by gas chromatography with flame photometric detection. Chemosphere. 68, 1–9. Willemsen, F.; Wegener, J.W.; Morabito, R.; Pannier, F. (2004) OT-SAFE. Sources, consumer exposure, and risks of organotin contamination in seafood. Final report of the European Commission Research Project OT-SAFE N QLK1–2001-01437. Xiao, Q.; Hu, B.; He, M. (2008) Speciation of butyltin compounds in environmental and biological samples using headspace single drop microextraction coupled with gas chromatography-inductively coupled mass spectrometry. J Chromatogr A. 1211, 135–141. Zachariadis, G.A.; Rosenberg, E. (2009) Speciation of organotin compounds in urine by GC-MIP-AED and GC-MS after ethylation and liquid-liquid extraction. J Chromatogr B. 877, 1140–1144. Zhong, Z.; Yang, M.J.; Pawliszyn, J. (1994) Solid-phase microextraction. A solvent-free alternative for sample preparation. Anal Chem. 66, 844A-853A. Zuliani, T.; Lespes, G.; Milacˇicˇ, R.; Šcˇancˇar, J.; PotinGautier, M. (2006) Influence of the soil matrices on the analysis performance of headspace solid-phase microextraction for organotin analysis by gas chromatography-pulsed flame photometric detection. J Chromatogr A. 1132, 234–240. Zuliani, T.; Lespes, G.; Milacˇicˇ, R.; Šcˇancˇar, J.; PotinGautier, M. (2008) Comprehensive study of the parameters influencing the detection of organotin compounds by a pulsed flame photometric detector in sewage sludge. J Chromatogr A. 1188, 281–285.

Chapter 12 Determination of Heavy Metals in Food by Atomic Spectroscopy Joseph Sneddon

Introduction In a developing society with an increasing demand for food (based on an increasing world population), the use of chemicals in the food industry (in animals, crops, and vegetables) has become universal due to such benefits as preserving; enhancing flavor, color, and presentation; reducing or minimizing pests; or simply to grow better and more productive food (greater volume from crops or increase mass of animals). The chemicals could have hormone-disrupting properties and may interfere with the normal action of hormones in animals and ultimately in humans in the food chain cycle. Generally speaking, organic compounds are considered to be the most likely endocrinedisrupting compounds, but heavy metals abound in the environment (water, air, plants, grasses, and soils) and can easily enter the food chain. The role of metals in the food chain is a complex area beyond the scope of this chapter. The heavy metal cadmium was shown to mimic the effects of estradiol in estrogenresponsive breast cancer cell lines (Stoica et al. 2000). Experimental data in vitro in animals show effects of cadmium on the hypothalamus-pituitary axis at different levels. This may lead to disorders of the endo-

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

crine and/or immune system (Schoeters et al. 2008). Different independent studies indicated that environmental exposure to Pb leads to delay in growth and pubertal development in girls (Dyer 2007). Pb affects hormones and responsiveness of all levels of the hypothalamic-pituitary-ovarian-axis. Exposure to Pb causes altered and delayed spermatogenesis accompanied by decreased fertility (Dyer 2007). Exposure to Hg results in menstrual disorders, subfecundity, and adverse pregnancy outcomes (Dyer 2007). Further information on endocrine disruption of heavy metals can be found in (Hontela and Lacroix 2006). There are numerous analytical techniques for metal determination, such as various electrochemical methods of voltammetry and coulometry and neutron activation analysis, etc., but this chapter will discuss only atomic spectroscopic techniques. These are the most widely used techniques and are accepted for the determination of metals in food.

Atomic spectroscopy methods Interaction of energy (light) with matter (gaseous atoms) produces three closely related, yet separate atomic phenomena, namely atomic absorption, atomic emission, and atomic fluorescence. In these techniques, the atoms are detected by optical means. A closely related technique is that of plasma source-mass spectrometry, in particular, 289

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inductively coupled plasma-mass spectrometry. In this technique, the atoms are detected by mass spectrometry. These techniques are collectively known as atomic spectroscopy or spectrochemical techniques and have detection limits for metals and metalloids ranging from micrograms per milliliter to nanograms per milliliter and even as low as picograms per milliliter in solutions (micrograms per gram to as low as picograms per gram in solid samples). These techniques have been used to detect metals and metalloids in solids, liquids, and gasses in just about every conceivable matrix, including biological, clinical, environmental, pharmaceutical, and in petroleum products as well as food. Although all metals are (or may be) important in food and food products, the most commonly determined are cadmium (Cd), mercury (Hg), and lead (Pb). These have no known value or need in the human body and are frequently introduced into the environment and consequently the food chain from industrial (accidents) sources. Historically, atomic spectroscopic techniques determined total metal concentration, but interest in the species or oxidation states of metals has grown rapidly in the last few years. Although this interest was driven by the clinical or biochemical users who recognized the importance of species of metals in human health issues, it is safe to say that the food industry is also interested in species of metal. Most common determinations of species involve a hyphenated method, typically separations (various chromatography techniques) followed by atomic spectroscopic detection (atomic absorption spectrometry, atomic emission spectrometry, atomic fluorescence spectrometry [AFS], and inductively coupled plasma-mass spectrometry) or a somewhat complex extraction procedure to separate species prior to determination by atomic spectroscopic techniques. The object of this chapter is to give the reader an overview of atomic spectroscopy techniques, including instrumentation and general analytical performance. It is not intended to be comprehensive or discuss

many areas on the fringe of atomic spectroscopy. It is beyond the scope of this chapter to describe in detail these techniques, and the reader is referred to a number of texts that provide detailed discussion of these four analytical phenomena (Ingle and Crouch 1988; Lajunen 1992; Haswell 1991; Nolte 2003). Much of this material, at least the first part of this chapter, has been reported recently by Sneddon and Thibodeaux (2009). This chapter provides an overview (basic theory and instrumentation) of atomic absorption spectrometry (AAS), atomic emission spectrometry (AES) with an inductively coupled plasma as the excitation source, atomic fluorescence spectrometry (AFS), and inductively coupled plasma-mass spectrometry (ICP-MS). Additional atomic spectroscopic techniques will be described, namely cold vapor-atomic spectroscopy (this has extensive use in Hg determination in seafood) and hydride generation-atomic spectroscopy (HG-AS) (used in food industry). Some information on the practice of atomic spectroscopy as well as the very important area of sample preparation of food prior to atomic spectroscopic analysis is presented.

Atomic absorption spectrometry Theory Atomic absorption (AA) involves the impingement of light of a specific wavelength onto gaseous atoms. This causes a valence electron in the atom to be raised from a lower energy level to a higher energy level (called an electronic transition). When the energy of the photon is identical to the energy difference between the lower and higher energy level of the atom, then absorption will occur. The intensity of this transition is related to the original concentration of the ground state atoms. This can be represented as follows: T = I Io

(12.1)

where T is the transmittance, I is the intensity of the light source passing through the sample

Determination of Heavy Metals in Food by Atomic Spectroscopy

zone, and Io is the intensity of the light source before it passes through the sample zone. The sample zone or path length, b, is relatively long to maximize the amount of light absorbed by the atoms. The amount of light absorbed will depend on the atomic absorption coefficient, k. This value is related to the number of atoms per cubic centimeter in the atom cell, n; the Einstein probability for the absorption process; and the energy difference between the two levels of the transition. In practice, these are all constants that are combined to give one constant, called the absorptivity, a. The coefficient k is related exponentially to the transmittance as follows: T = I I o = e − kb

(12.2)

In practice, the absorbance, A, is used in AAS and is related logarithmically to the transmittance as follows: A = − log T = − log I I o = log I o I = log 1 T = kb log e = 0.43 kb

(12.3)

The Beer-Lambert law relates A to the concentration of the metal in the atom cell, c, as follows: A = abc or

A = eo bc

(12.4)

where a is the absorptivity in gram per litercentimeter, eo is the molar absorptivity in gram per molar-centimeter, and b is the atom cell width in centimeters. AAS involves the measurement of the drop in light intensity of Io to I (depending on the concentration of the metal). Current and modern instrumentation automatically converts the logarithmic value into A. Absorbance is a unitless number, typically 0.01 to 2.0. In practice, it is better to work in the middle of this range (recommended 0.1–0.3 A) because the precision is poorer at the extremes due to instrumental noise. The most intense transition from the ground state to the first excited state (resonance transition) is the most widely used transition because it is the most sensitive.

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The origins of atomic spectra and detailed discussion are available elsewhere (Sobelman 1992). Atomic absorption spectrometry (AAS) was discovered independently by Walsh (1955) and Alkemade and Milatz (1955). Instrumentation A basic AA system consists of six basic parts: a light source, an atomizer, a sample introduction system, a wavelength-selection device, a detection system, and a readout system. All the components are conveniently packaged in a complete benchtop unit and are connected to a computer for control, sample preparation, data reduction, and printout. There are numerous commercial instrumentation available, with costs ranging from a small compact flame AAS for around $10,000, to a top-of-the line multi-metal flame/furnace AAS system with automatic sample introduction and data station for around $100,000. A detailed description of light sources (hollow cathode lamps and electrodeless discharge lamps), wavelength selection devices (monochromators), sample introduction systems (pneumatic nebulizers), detection systems (photomultiplier tubes), and readout (connected to external computers) are described elsewhere (Ingle and Crouch 1988; Lajunen 1992; Haswell 1991; Nolte 2003). Sample introduction is very important in AAS (and AES and ICP-MS) and is discussed in detail elsewhere (Sneddon 1990). A short discussion on the atomizer is included in this chapter. Atomizer The only widely used and accepted atomizers in AAS are the flame and graphite furnace. In flame AAS, the sample is (usually) introduced into the flame as a fine mist or aerosol. Flames consist of an oxidant and a fuel. The most widely used flames in AAS are air-acetylene (air is the oxidant and acetylene the fuel) and nitrous oxide-acetylene (nitrous oxide is the oxidant and acetylene the fuel).

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Analysis of Endocrine Disrupting Compounds in Food

These flames are called combustion flames. Other flames, called diffusion flames, have been proposed but are not widely used. The primary object of the flame is to dissociate the molecules into atoms. Air-acetylene (2500 K) does this readily and efficiently for about 40 to 50 metals in the periodic table. The other 10 to 20 metals in the periodic table require the hotter nitrous oxide-acetylene flame (3200 K). A long, thin flame is desirable in AAS for maximum sensitivity. The graphite furnace atom cell or electrothermal atomizer (ETA) for AAS was commercially developed in the late 1960s. Its principal advantages over flame atomizers are the improvement in sensitivity, typically 10 to 100 times; the ability to use microvolumes (2–200 μL) and micro-mass solids (few milligrams) sampling; and in situ pretreatment of the sample. However, ETAs are prone to interference, particularly from alkali and alkaline earth halides, and require a more complex (and subsequently more expensive) system. The use of an electrically heated tubular furnace was first reported by King in 1905, but for analytical chemistry the work and system developed by L’Vov (1961) is regarded as the forerunner of present day ETAs. It consisted of a carbon electrode in which the sample was applied and a carbon tube that could be heated by electrical resistance. The initial design used a supplementary electrode for preheating the furnace, lined the carbon tube with tungsten or tantalum foil to minimize vapor diffusion, and purged the system with argon to prevent oxidation of the carbon. Later work involved direct heating of the sampling electrode by resistance heating, and the tube was made of pyrolitic carbon. After heating the tube to an elevated temperature, the sample electrode was inserted into the underside of the tube, and vaporization of the sample was confined to the tube where AAS measurements were made. The system was difficult to operate, and the reproducibility could be poor.

In 1968, Massmann described a heated graphite atomizer (HGA), which was commercially developed by the Perkin-Elmer Corporation and proved to be the forerunner for all current commercial ETAs. An isothermal type furnace system proposed by Woodriff (1974) at around the same time was considered more difficult to commercialize, although recent work has shown the advantage of atomization under isothermal conditions. The Massmann system was typically a 50-mm-long and 10-mm-diameter graphite tube that was heated by electrical resistance, typically 7–10 volts at 400 amps. An inert gas, usually argon or nitrogen, flowed at a constant rate of around 1.5 L/min, and the entire system was enclosed in a water jacket. A microliter sample was deposited through an entry or injection port in the center of the tube and could be heated in three stages by applying variable current to the system; drying to remove the solvent, ashing or pyrolysis to remove the matrix, and finally atomization of the element. Careful control of the temperature was required in order to obtain good reproducibility. In 1969, West and Williams developed a rod or filament atomizer. It consisted of a graphite filament of 40 mm in length and 2 mm diameter, supported by water-cooled electrodes, and heated very quickly by using current of 70 amps at 10–12 volts. Shielding from the air was achieved by a flow of inert gas around the filament. Although primarily developed for AAS, West and Williams showed the potential of the system for AFS. The West filament was the forerunner for the mini-Massmann atomizer developed commercially by Varian Associates. A commercial system was called the carbon rod atomizer (CRA 63). Its main advantages were in its somewhat simpler and less-complex design as compared to the HGA, low power requirements (2–3 kilowatts), and fast (∼2 s) heating rate. There were differences between this system and the West filament, principally the drilling of a hole in a solid cylindrical

Determination of Heavy Metals in Food by Atomic Spectroscopy

graphite tube and later using a small cup or crucible between two spring-loaded graphite rods. The system was proposed for low microliter volumes, typically 1–20 μL. In general, detection limits and increased interferences were found using the CRA type system compared to the HGA, and this type of system was discontinued around the mid-1980s and is not currently available commercially. Graphite furnace atomic absorption spectrometry has essentially the same instrumentation as flame AAS, except for the (a) atom cell and (b) sample introduction system. An additional need in furnace AAS is faster electronics to process the transient and faster generated signal compared to flame AAS. In practice, AAS usually has fast electronics capability and most commercial systems use the flame and furnace interchangeably. Typical volumes used with a graphite furnace are from 1 to 200 μL. This volume can be conveniently introduced to the furnace manually by using a micropipette. There are various dedicated micropipettes available for this volume range, as well as adjustable micropipettes. In the late 1970s, it was suggested that the precision of furnace AAS could be improved by automatic introduction systems. This led to systems that could be added to furnace AAS for automatic sample introduction. It was shown that the precision was not significantly improved by automatic sample introduction, but nevertheless it is now an accepted part of furnace AAS, particularly for unattended operation and when numerous samples are required to be analyzed. These systems can be incorporated into sample preparation stations. A monograph by Butcher and Sneddon (1997) provides an in-depth coverage of areas in GFAAS (graphite furnace atomic absorption spectrometry), such as matrix or chemical modification, to allow a higher ashing temperature without loss of the more volatile metals such as Cd; background correction techniques, in particularly various configurations of the Zeeman effect (inverse ac longi-

293

tudinal and inverse transverse ac and dc) for a more accurate measurement; graphite tube design and material that allows a more rapid heating of the furnace to improve the signal from more volatile metals; and platform atomization Atomic absorption spectrometry reached maturity in the mid-1990s since its initial development in the mid-1950s. Current developments are in refinements and modest improvements, for example, software.

Atomic emission spectrometry Theory Atomic emission spectrometry (AES) involves the impingement of an external source of energy on ground state atoms. The radiation from these atoms is what is observed in AES. The probability of transitions from a given energy level of a fixed atomic population was expressed by Einstein in the form of three coefficients, termed transition probabilities, Aji (spontaneous emission), Bij (spontaneous absorption), and Bji (stimulated emission), which can be considered to represent the ratio of the number of atoms undergoing a transition to an upper level to the number of atoms in the initial or lower level. It can be represented as follows: N j = No

gi exp ( − Δ E KT ) go

(12.5)

where No is the number of atoms in the lower state (or ground state usually for analytical work), Nj is the number of atoms in the excited or upper level, gj and go are the statistical weights of the jth (upper state) and o (ground state), ΔE is the difference in energy in joules between these two states, K is the Boltzmann constant (1.38066 × 10−23 J/K), and T is the absolute temperature. If self-absorption is neglected, then the intensity of emission, Iem is I em = A ji hν ji N j

(12.6)

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Analysis of Endocrine Disrupting Compounds in Food

where h is Planck’s constant (6.624 × 10−24 Js), and νji is the frequency of the transition (ΔE = h ν). Therefore, N is directly related to the concentration of the solutions as follows: Nj = N

gi exp ( − ΔE KT ) go

(12.7)

The intensity emission of a spontaneous emission line, Iem is related to this equation (sometimes called the Maxwell-Boltzmann equation) as follows: I em = A ji hν ji N

gi exp ( − Δ E KT ) go

(12.8)

It can be seen that the atomic emission intensity is dependent on temperature and wavelength. Thus, a higher temperature at longer wavelength would give the most intense atomic emission signal. A plot of emission intensity against sample concentration will be linear. Atomic emission spectrometry (and atomic fluorescence spectrometry) has linearity extending up to five to seven orders of magnitude compared to two to three orders of magnitude of AAS. Instrumentation The primary components of an atomic emission spectrometer (AES) are similar to those of AAS, although for optimum performance the components are different. The excitation source and atomization source are the same, most frequently a plasma. The higher temperature of the plasma will lead to a richer spectrum with many more lines. In order to separate these lines and prevent or minimize spectral interferences, a high-resolution monochromator is required. The 0.20 meter grating typically used in AAS does not provide the resolution required. The most widely adopted system is the Echelle Monochromator, which uses high diffraction orders and large angles of diffraction. Resolution is around 0.015 nm compared to

around 0.2 nm for a typical AAS monochromator. During the development of the plasma as an excitation source in analytical AES, PMTs (photomultiplier tubes) were first used. However, they have very limited use in multichannel systems, and AES quickly adopted systems capable of multimetal analysis. In the early 1980s, the photodiode arrays (PDAs) and image-sensing vacuum tubes, the socalled Vidicons, were used. A PDA consists of arrays (256, 512, 1024, and 2048 elements arranged in linear manner) of photodiodes operated on a charge transfer storage mode. Each diode is sequentially integrated (several microseconds) after all the diodes have been integrated with the incident radiation. The current generated by each photodiode is proportional to the intensity of the radiation it receives. The sequential measurement of the current can occur many times a second under the control of a microprocessor. This digitized information can be stored in a computer for electronic processing and visual display. Diode array systems are excellent for studying transient signals such as those on the laser-induced plasma with gate-delay generator systems. However, it does have somewhat of a limited resolution, usually 1–2 nm. Diode arrays are used in Vidicons in the form of a spectrum. These are similar to a small television tube. From the late 1980s to the present, interest has been shown in the use of the charge transfer device (CTD), specifically the chargecoupled device (CCD), and to a lesser extent the charge-injection device (CID). These are solid-state sensors that have integrated circuits. The charge generated by a photon is collected and stored in a capacitor. A typical pixel arrangement can be 512 × 320 CCD (much larger arrangements such as 2000 × 2000 pixels have been constructed). The capacitor can be reversed, biased by a positive voltage applied to the electrode, creating a potential well. The photons striking the array give electron-hole pairs, and the

Determination of Heavy Metals in Food by Atomic Spectroscopy

MASS SPECTROMETER

Ion Detector

Signal Handling

Quadrupole Mass Flier

Turbomolecular Pump

PLASMA SAMPLING IINTERFACE

Lan Lenses Skimmer including Cone Portion Slap

Turbomolecular Pump

INDUCTIVELY COUPLED PLASMA

Sampling Cone

Rotary Pump

Plasma Torch

295

SAMPLE INJECTION SYSTEM

Nebulizer

ETV

RF induction Coil

Sample

RF Power

Argon

optional

FIA-HG

Laser Probe

optional

Data processing

Figure 12.1. Typical quadruple ICP-MS system with various sample introduction options (ETV, electrothermal vaporization; LA, laser probe or ablation; FIA-HG, flow injection analysis-hydride generation).

electrons can be stored for a short time in the well. The amount of charge accumulated is a direct function of the incident radiation (and time) and is very linear. The charge is shifted horizontally and down to a readout preamplifier that results in a scan of each row in series. CCDs are very useful when low levels of radiation are to be detected. At high levels, “blooming” occurs, which results in curvature of the response. A CID is a twodimensional array of pixels. The photons generate positive charges below the negative well capacitors. Again, the amount of charge is proportional to the incident radiation. There is a rapid development of the CTD in spectrochemical analysis. The development of the inductively coupled plasma (ICP) as an excitation source for analytical atomic emission spectrometry has been a major advancement in atomic spectroscopy. Its higher temperature has made this source the choice for many atomic spectroscopists’ work. The most common plasma source is the inductively coupled plasma (ICP), which was first developed in the mid-1960s by Fassel and coworkers at Iowa State University and Greenfield and coworkers at Albright and

Wilson Ltd. in England. It became commercially available around the mid-1970s. A typical ICP is shown in Figure 12.1 and consists of three concentric quartz tubes. These are frequently referred to as the outer, intermediate, and inner or carrier gas tubes. The outer tube can have various sizes in the range of 9–27 mm. A two- or three-turn induction coil surrounds the top of the quartz tube or torch and is connected to a radiofrequency generator. The coil is water cooled. The argon, typically at a flow rate of about 1–2 L/ min, is introduced into the torch, and the radio frequency field is operated at 4–50 MHz, most typically 27.12 MHz, and a forward power of 1–5 kW, typically 1.3 kW, is applied. An intense magnetic field around the coil is developed, and a spark from a Tesla coil is used to produce “seed” electrons and ions in this region. This induced current flowing in a closed circular path results in great heating of the argon gas, and an avalanche of ions is produced. Temperatures in an ICP have been estimated to be around 8000–10,000 K. The high temperatures necessitate cooling, which is applied using argon to the outer tubes at flow rates as high as 17 L/min. The sample is introduced, usually as an aerosol, through the

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Analysis of Endocrine Disrupting Compounds in Food

inner tube and is viewed at a distance of 5–20 mm above the coil. The advantages of the ICP include high temperature, long residence times, presence of no or few molecular species, optical thiness, and few ionization interferences. The last decade has seen a tremendous amount of effort evaluating and understanding the ICP through numerous studies on mechanisms and characterizing variations of the system. The reader is referred to a recent book edited by Montaser and Golightly (1992) that describes the current status of the ICP. Considerable improvement and refinement in plasma source AES has occurred over the last decade. Improved detection limits have been achieved by rotating the plasma through 90 degrees and by the development of the miniature ICP. Considerable effort has been expended in the area of sample introduction (see earlier discussion). Improved software has pushed ICP-AES into a well-established and frequently used technique, particularly for multimetal AES.

Atomic fluorescence spectrometry Theory Atomic fluorescence spectrometry (AFS) has been around for about 100 years and has attracted a good deal of interest as an analytical technique from the early 1960s through early 1980s. From that time until recently it became a lesser used atomic spectroscopic technique. The availability of modest-cost commercial instrumentation, most notably from China and Canada, has meant a rise in its use. However, this author does not see it as a major player (as yet) in the atomic spectroscopic technique area. The only practical (with sufficient sensitivity) AFS transition used for quantitative work is resonance fluorescence. A certain sequence of events produce resonance AFS. Production of atoms in an atomizer is followed by elevation of these ground state

atoms to the excited state through absorption by incident radiation. Radiation from the excited state back to the ground state yields resonance AFS. The absorbed and emitted radiation are equal for resonance AFS. There are slight differences between narrow line sources (e.g., hollow cathode lamps and electrodeless discharge lamps) operated at a low current of a few milliamperes and continuum sources (xenon arc or quartz halogen lamps). These differences are due to different source atomic absorption profiles. In this chapter we will discuss only AFS with a line source because it is the most common commercial system in use at present. The radiance of atomic fluorescence (AF), BF, (in ergs s−1 cm−2 sr−1) emitted at right angles from a totally illuminated, parallel piped gas containing a uniform distribution of atoms can be expressed as BF = K n f δ Bs Ψ Ω ( L l1 As )

(12.9)

where K is the atomic absorption coefficient for Doppler broadening (nm cm2 s−1) n is the number of atoms in lower-level absorption transition (cm−3) f is the oscillator strength (no units) δ is the factor to allow finite width of line source compared with absorption line (no units) Bs is the radiance line source (ergs−1 cm−2 sr−1) Ψ is fluorescence power yield (ergs fluoresced per second/ergs absorbed per second) Ω is the solid angle of source radiation reaching the atom cell (sr) L l1 is the fluorescence cell dimensions (cm3) As is the surface area of fluorescence cell (cm2) Because AF is isotropic, a further factor, Ωf, must be added to obtain the AF signal observed by a detection system. Here, Ωf is the solid angle (sr) oscillated by the detecting

Determination of Heavy Metals in Food by Atomic Spectroscopy

optics. Inspection of equation (12.9) shows a direct proportionality between AF radiance (Bf) and the concentration of the ground state atoms. A further proportionality indicated by equation (12.9) lies between the fluorescence radiation (Bf) and the radiation of the line source (Bs). It can therefore be concluded that excellent sensitivities can be achieved by high-intensity light sources. This relationship was the driving force in AFS in the early 1960s through the late 1970s, with considerable effort by numerous workers to obtain high-intensity line sources (and continuum sources, for that matter). However, despite some promise, particularly for selected metals using microwave-excited electrodeless discharge lamps (EDLs) and the laser, highintensity light sources for AFS were never realized, and the technique never reached or fulfilled its potential (in terms of sensitivity). Current commercial AFS systems use intensity line sources but at nowhere near the high intensity needed for low sensitivity. Instrumentation A typical AFS system consists of parts similar to those used in atomic absorption (AA) and atomic emission (AE). In fact it is almost a combination of these two techniques: a light source, atomizer, sample introduction system, wavelength selection device, detection system, and readout system. All components are conveniently packaged into a similar (AA and AE) benchtop arrangement and connected to various computer or data reduction stations. These components are discussed above. Unique to AFS is that the light source and detection system are at right angles to each other to maximize the AF signal and reduce scatter (an interference).

Inductively coupled plasma mass spectrometry Since the early to mid-1980s, inductively coupled plasma (ICP) has been used as the

297

ion source for mass spectrometry to determine metals. Its advantages include from two to three orders of magnitude improvement in sensitivity compared to traditional ICP-AES; the mass spectra of the metal are very simple and unique, giving high specificity and inherent multimetal coverage; and the technique will measure metal isotopic ratios. Disadvantages include potential spectral interferences from molecular species and the increased cost and complexity of instrumentation. An inductively coupled plasma-mass spectroscopy (ICP-MS) system consists of the ion source, which is the ICP, an interface system that consists of a sampling cone, a differentially pumped zone and a skimming zone, ion lenses, a quadrupole mass spectrometer, and a detector. A schematic diagram of a typical ICP-MS is shown in Figure 12.1. A detailed description of the instrumentation and the performance of ICP-MS is described elsewhere (Nolte 2003; Montaser and Golightly 1992). Essentially, the ICP is in a horizontal position and works under atmospheric or normal pressure. Ions produced by this ICP are introduced to the MS through a small orifice, typically 1 mm diameter. The MS is at low pressure, typically at 10−5 to 10−6 torr. ICP-MS has increasingly become the choice of many spectrochemical analysts.

Cold vapor atomic spectroscopy Mercury’s unique properties, high toxicity, and common use in many industrial processes has led to the development of the cold vapor accessory (CV). It has been applied to all atomic spectroscopy techniques (AAS, AES, AFS, and ICP-MS). Mercury has an appreciable vapor pressure at room temperature (0.16 Pa at 25°C). Mercury (in the ionic form) and in an acidic medium can be reduced by stannous chloride to produce ground-state atomic mercury. After equilibration, the mercury vapor is swept from the reaction

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vessel with a carrier gas (argon, air, or nitrogen) into the optical path of an atomic absorption instrument for determination as a transient signal. Alternatively, a closed system will produce a steady-state signal. The primary advantage of CV-AS for Hg determination is a low detection limit of submicrograms per milliliter. This can be lowered further using a dedicated commercial cold vapor atomic fluorescence spectrometry system (CV-AFS), where a detection limit in tens of nanograms per milliliter is possible. It should be noted that type and concentration of acid, chemical form (inorganic versus organic mercury), matrix components, and other components, such as the reducing agents, can degrade these low-detection limits. The use of stannous chloride as a reducing agent will not reduce organomercury compounds. Various pretreatment processes have been developed to overcome this potential problem.

Hydride generation-atomic spectroscopy Hydride generation (HG) has been around for well over 100 years and used for the determination of arsenic in such methods as the Marsh reaction and Gutzeit test. It has been successfully incorporated and applied in all branches of atomic spectroscopy (AAS, AES, AFS, and ICP-MS). Its popularity arises from its relative simplicity and low cost, but gives significantly increased sensitivity, typically 1 μg/mL or lower. It is limited to eight metals, As, Bi, Ge, Pb, Se, Sb, Sn, and Te. Typically, a solution containing the hydride-forming metal is reacted most commonly with a sodium borohydride in an acidic medium to form the gaseous hydride that is then swept or moved to the atomizer for atomic spectroscopic detection. An example would be 3 BH −4 + 3 H + + 4 H 3 AsO3 = 3 H 3 BO3 + 4 AsH 3 + 3 H 2 O The reader is referred to a monograph in this area (Dedina and Tsalev 1995), which deals

with many aspects of hydride generation including including theory and fundamentals, hydride release and transport, atomizers, interferences, and numerous methodologies and applications. Although this monograph was published in 1995, HG-AS continues to be an active research area to further understand the mechanism and application and in the area of speciation.

Practice of analytical atomic spectroscopy The choice of which analytical atomic spectroscopic technique to use will depend on the analysts’ needs and expectations, as well as the sample. There are many and varied commercial systems available, or the analyst may decide that the sample is best suited to a laboratory-constructed system. The size of sample, whether it is a solid, liquid, or gas; the level to be detected; the accuracy and precision that is acceptable; availability of a particular system; cost per sample; and the speed of analyses are factors to be considered. Atomic spectroscopic methods are techniques that depend on the comparison of signals obtained from samples with those obtained from sample standards of known composition. In most cases, these standards are aqueous solutions of the metals of interest. However, the analysis of real samples is complicated by the fact that the metal of interest is present as part of a sample matrix. The matrix can cause an interference in the analysis. Therefore, in analytical atomic spectroscopy, much attention is paid to the possibility of interference. This can lead to reduced or poor accuracy. Accuracy can be defined as how close the atomic spectroscopic analysis is to the “correct” answer. In a typical method development, accuracy will be established in many ways, including standard additions, comparison of the results of the atomic spectroscopic analyses with the results from a different method, recoveries or spikes, or

Determination of Heavy Metals in Food by Atomic Spectroscopy

applying the atomic spectroscopic method to standard samples such as those supplied by the National Institutes of Science & Technology (NIST) (Gaithersberg, MD). A concern of analytical atomic spectroscopy is precision, which can be defined as the repetitive analyses of a particular sample expressed as a percentage. Precision will vary with many factors, including the sample, the level to be determined, and the choice of instrumentation. Finally, the detection limit is an important factor in analytical atomic spectroscopy. Current atomic spectroscopic techniques have solution detection limits in the microgram per liter to microgram per milliliter range. However, lower detection limits are possible using newer and improved techniques in analytical atomic spectroscopy. The reader is referred to a recent book by Butcher and Sneddon (1997), which describes the practice of graphite furnace atomic absorption spectrometry. Much of the advice and suggestions in the book could be equally applied to many areas of analytical atomic spectroscopy.

Sample preparation for metal determination in food by atomic spectroscopic methods Most analyses are preferentially performed on solution samples. This can be attributed to the desire for a more homogeneous analysis sample and concern over whether a few micrograms or milligrams will be representative of the bulk properties of a large solid sample. Also, improved precision and accuracy is frequently obtained with solution samples (as opposed to solid samples), and most commercial instrumentation performs at an optimum with solution samples. Therefore, sample preparation still remains an integral part of spectrochemical analysis and is widely used in the preparation of seafood for metal determination by spectrochemical techniques. However, although the metal

299

determination is most frequently performed on solutions, food results are most commonly reported as micrograms per gram or in some cases as nanograms per gram. Furthermore, results from metals in food studies correlated with humans are often presented as milligrams per kilogram or micrograms per kilogram of mass. Sample preparation can be conveniently divided into two areas, classical and microwave. Classical methods involve wet or acid decomposition and the use of various mineral acids (HNO3, H2SO4, HClO4, and/or HF) and oxidizing agents (typically H2O2) to effect dissolution of the sample. It can be performed on an open or closed system. Microwave digestion has rapidly become the choice for many digestion/dissolutions, particularly in seafood preparation (Kingston and Haswell 1997). It involves the use of 2450-Mhz electromagnetic radiation to digest samples in a Teflon or quartz container. Commercial systems are readily available, are conveniently automated, and can digest up to 48 samples simultaneously under controlled temperature conditions. A recent review describes sample preparation on spectrochemical samples for solid materials (see Sneddon et al. 2006).

Selected applications of determination of metals in food In the following section, selected applications of atomic spectroscopy applied to metal determination in various food are presented. Previously, selected applications of atomic spectroscopic methods for determination of metals in seafood have been discussed and the reader is referred to these references (Sneddon and Thibodeaux 2009; Sneddon et al. 2007; Sneddon 2007). Seafood is an important area, but it is not discussed in this selected application. Not included in this report are metals in wine. Metals in wine are often determined to pinpoint the geographical location of the wine (grape and soil), and often the quality is

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Analysis of Endocrine Disrupting Compounds in Food

Table 12.1. Selected results of metals in food. Metals

Samples Analyzed

Method

Mn and Cr

Meats, dairy products, vegetables, and fruits

Tinggi et al. 1997

Cr Zn, Cu, Cd, Pb, Ni, and Cr Hg, As, Fe, Cu, Zn, Pb, Cd, and Ni Pb, Cd, Cu, Cr, Co, Ni, Mn, and Zn Cd, Pb, and As

Fruits and vegetables Cabbage, wheat, potato, instant milk, fish, eggs, and baby foodstuffs Chocolate

Flame AAS and GFAAS Flame AAS Flame AAS and ETAAS Flame AAS and GFAAS ICP-AES ICP-MS and GFAAS ICP-AES ICP-MS

Jorhem et al. 2008 Rubio et al. 2009 Perello et al. 2008

Fe, Cu, Mn, Zn As, Cd, Hg, and Pb

Honey and sugars Food additives and contaminants

References

Soceanu et al. 2008 Milacic and Kralj 2008 Guldas et al. 2008 Ioannidou et al. 2005

ICP-MS

Julshamn et al. 2007

Sb(III) and Sb(V) Hg Hg

Cereals Fish (sardine, hake, and tuna), meat (veal steak, loin of pork, breast and thigh of chicken, and steak and rib of lamb), string beans, potato, rice, and olive oil. Carrot puree, fish muscle, mushroom, graham flour, simulated diet, scampi, and mussel powder Mushrooms Cereals, fruits, and vegetables Milk

HG AFS CV-AAS CV-AFS

Hg

Vegetables

CV-AFS

Ferrieira et al. 2009 Jedrzejczak 2002 Cava-Montesinos et al. 2004 Ma and Xiangli 2007

As, Cd, Hg, and Pb

AAS, atomic absorption spectrometry; AFS, atomic fluorescence spectrometry; CV, cold vapor; GFAAS, graphite furnace atomic absorption spectrometry; HG, hydride generation; ICP-AES, inductively coupled plasma-atomic emission spectroscopy; ICP-MS, inductively coupled plasma-mass spectrometry; ICP-OES, inductively coupled plasmaoptical emission spectroscopy.

determined by the concentration of selected metals (and organics). The results are summarized in Table 12.1 and described in more detail in the cited references. This section is not meant to be comprehensive but rather to show numerous results of several studies involving various food atomic spectroscopy techniques and the various metals determined. Tinggi et al. (1997) investigated several wet digestion procedures using various acid mixtures of HNO3/H2SO4/HClO4 and HNO3/ H2SO4 for the decomposition of meats, dairy products, vegetables, and fruits prior to determination of manganese and chromium by flame AAS and Zeeman graphite furnace atomic absorption spectrometry (GFAAS). The addition of hydrofluoric acid (HF) to the mixture of HNO3/H2SO4 was also investigated for determination of Cr. All the acid mixtures tested were found to be satisfactory, but for reasons of safety, HNO3/H2SO4 was

the method of choice. Analysis of the food samples found relatively high levels of manganese and chromium in most cereal products, with dairy products (except for Cr in cheese), vegetables, and fruits containing relatively low levels of both metals. Chromium concentrations were determined by flame AAS in vegetables and fruits grown in urban and rural areas to establish the potential human exposure through food chains by Soceanu and others (2008). Chromium concentrations in vegetable samples were between 0.06 and 2.16 mg/kg and in fruit samples they ranged from less than the detection limit to 0.95 mg/kg. An acid-assisted microwave digestion procedure was developed for the determination of Zn, Cu, Cd, Pb, Ni, and Cr in Slovenian foodstuffs (cabbage, wheat, potato, instant milk, fish, eggs, and baby foodstuffs) using flame AAS and ETAAS (Milacic and Kralj

Determination of Heavy Metals in Food by Atomic Spectroscopy

2008). The repeatability of measurements was tested in the cabbage sample and was ±3.3% for Zn, ±4.6% for Cd, ±6.8% for Cu, and ±15.5% for Cr. The worse value obtained for Cr was probably due to its inhomogeneous distribution and very low concentration in the cabbage. Concentrations of Ni and Pb in the cabbage sample were below the limit of detection (0.2 mg/kg). The concentrations of Zn, Cu, Cd, Pb, Ni, and Cr found in cabbage, wheat, and potato were comparable to the literature data. Cadmium, Pb, and Ni were not found to be the contaminants in the foodstuffs analyzed. Higher concentrations of Ni were found in the baby food only due to the cocoa that the baby food contained. The Cr content was very low in baby food, yolk and white of eggs, and fish (<0.05 mg/kg). Different types of chocolate (plain chocolate, milk chocolate, and chocolate with pistachio) were supplied from different markets in Bursa, Turkey, and were tested for Hg, As, Fe, Cu, and Zn using FAAS and for Pb, Cd, and Ni using GFAAS by Guldas and others (2008). According to chocolate types, contents ranged from (in milligrams per kilogram) 0.03 to 0.04 (Pb), 0.02 to 0.04 (Hg), 0.02 to 0.03 (Cd), 0.01 to 0.02 (As), 1.29 to 4.78 (Ni), 5.74 to 10.61 (Cu), 2.52 to 3.67 (Fe), and 12.11 to 16.68 (Zn). The levels of Cu, Zn, and Fe were highest in the samples of chocolate with pistachio. The contents of Hg and Ni were highest in milk and plain chocolates, respectively. Arsenic was highest in two chocolate types (milk chocolate and chocolate with pistachio). There was no difference among the chocolate types in terms of Pb and Cd contents (p < 0.01). The heavy metal contents of the analyzed samples were within the ranges reported by the World Health Organization and Turkish Food Codex regarding chocolate and other foods. A method for the determination of metals in honey and sugars without prior digestion or ashing of the sample was developed using ICP-AES by Ioannidou et al. (2005). The sensitivity was investigated using calibration

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curves obtained in presence of honey and sugar matrices. The obtained recoveries for Cd, Cu, Cr, Co, Ni, and Mn at the microgram per liter level were satisfactory and practically independent of the matrix used for the calibration standards. The recoveries of Pb and Zn were less sufficient. Various commercial samples of honey, sugar, glucose, and fructose were determined for the Pb, Cd, Cu, Cr, Co, Ni, Mn, and Zn concentrations. A survey of the levels of As (ICP-MS), Cd (ETAAS), and Pb (ETAAS) in different types of rice available on the Swedish retail market was performed in 2001–2003 (Jorhem et al. 2008). The types of rice included long- and short-grain, brown, white, and parboiled white rice. The mean levels found were as follows: total As, 0.20 mg/kg; inorganic As, 0.11 mg/kg; Cd, 0.024 mg/kg; and Pb, 0.004 mg/kg. In countries where rice is a staple food, it may represent a significant contribution in relation to the provisional tolerable weekly intake for Cd and inorganic As. To evaluate the daily dietary intake of essential metals in the Canary Islands, Spain, the Cu, Fe, Mn, and Zn concentration in 420 food and drink samples collected in local markets were determined by ICP-AES by Rubio and others (2009). The estimated daily dietary intakes of Cu, Fe, Zn, and Mn were 2.1 mg/day, 13.2 mg/day, 2.4 mg/day, and 9.0 mg/day, respectively. The Fe dietary intake was found to be below the recommendations fixed for adult women, and the Cu and Mn dietary intakes fulfilled the recommended dietary allowances. The mean daily intake of Zn was below the recommended dietary allowance. Cereals were found to be the food group that contributed most to the intake of these metals. Whereas the island of El-Hierro presented Cu, Fe, Mn, and Zn mean intakes over the estimated intakes for the whole archipelago, Fuerteventura Island showed the lowest intakes. Tenerife and Fuerteventura showed the lowest Fe intakes, being below the recommendations.

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The effects of cooking processes commonly used by the population of Catalonia (Spain) on As, Cd, Hg, and Pb concentrations in various foodstuffs were determined by ICP-MS by Perolla and others (2008). All food samples were collected at local markets. Foods included fish (sardine, hake, and tuna), meat (veal steak, loin of pork, breast and thigh of chicken, and steak and rib of lamb), string beans, potato, rice, and olive oil. For each food item, two composite samples were prepared for analyses for metal levels in raw and cooked (fried, grilled, roasted, and boiled) samples. The highest concentrations of As, Hg, and Pb (raw and cooked samples) were mainly found in fish, with a clear tendency, in general, to have increased metal concentrations after cooking. However, in these samples, Cd levels were very close to their detection limit. In turn, the concentrations of metals in raw and cooked meat were detected in all samples (As), or only in a very few samples (Cd, Hg, and Pb). A similar finding corresponded to string beans, rice, and olive oil, whereas in potatoes Hg could not be detected and Pb was detected only in the raw samples. In summary, the results of the present study show that, in general terms, the cooking process is only of a very limited value as a means of reducing metal concentrations. This hypothetical reduction depends upon cooking conditions (time, temperature, and medium of cooking). Thirteen separate laboratories participated in an collaborative study on a method for the determination of arsenic, cadmium, mercury, and lead by ICP-MS after pressure digestion, including a microwave heating technique (Julshamn et al. 2007). Their developed method was tested on a total of seven foods: carrot puree, fish muscle, mushroom, graham flour, simulated diet, scampi, and mussel powder. The metal concentrations in milligrams per kilogram in dry matter ranged from 0.06 to 21.4 for As, 0.03 to 28.3 for Cd, 0.04 to 0.6 for Hg, and 0.01 to 2.4 for Pb. The precision for As ranged from 3.8% to 24%,

for Cd from 2.6% to 6.9%, for Hg from 4.8% to 8.3%, and for Pb from 2.9% to 27%. The reproducibility relative standard deviations (RSDR) for As ranged from 9.0% to 28%, for Cd from 2.8% to 18%, for Hg from 9.9% to 24%, and for Pb from 8.0% to 50%. Hydride generation atomic fluorescence spectrometry (HG-AFS) was used for the determination of Sb(III) and Sb(V) in mushrooms by Ferriera and others (2009). The work concentrated on the efficiency of HG employing NaBH4, with and without a previous KI reduction. The extraction efficiency of total antimony and the stability of Sb(III) and Sb(V) in different extraction media (nitric, sulfuric, hydrochloric, acetic acid, methanol, and ethanol) was investigated with 0.5 mol/L. H2SO4 proved to be the best extracting solution for the speciation analysis of antimony in mushroom samples. The detection limit for both species was around 1 ng/g. The precision was 3.8% (14.7 ng/g) for Sb(V) and 5.1% (4.6 ng/g) for Sb(III). The recovery values obtained for Sb(III) and Sb(V) varied from 94% to 106% and from 98% to 105%, respectively. For five different mushroom samples the Sb(III) concentration varied from 4.6 to 11.4 ng/g, and Sb(V) varied from 14.7 to 21.2 ng/g. Total Hg concentrations were determined by CV-AAS in 573 samples of agricultural crops and foods of plant origin, including cereals, fruits, and vegetables, and their products from the Polish market (Jedrzejczak 2002). Mercury concentrations in the agricultural crops and plant foods were generally below the maximum permissible limits in Poland and rarely exceeded 5 μg/kg. Values ranged from <0.1 to 14 μg/kg, mean 2.4 ± 2.3 μg/kg in wheat and rye grains; from <0.1 to 2.4 μg/kg, mean 0.5 ± 0.4 μg/kg in nine varieties of vegetables; from <0.1 to 5.1 μg/kg, mean 1.1 ± 0.9 μg/kg in seven varieties of fruit; from <0.1 to 5.6 μg/kg in cereal products and jams; and from <0.1 to 3.0 μg/L in fruit and vegetable juices, nectars, and beverages. The contribution of the Hg in

Determination of Heavy Metals in Food by Atomic Spectroscopy

the analyzed agricultural crops and foods of plant origin to the weekly dietary intake of total Hg was 8 μg/person. A highly sensitive mechanized method was developed for the determination of mercury in milk by AFS by Cava-Montesinos and others (2004). Samples were sonicated for 10 min in an ultrasound water bath in the presence of 8% (v/v) aqua regia, 2% (v/v) antifoam A, and 1% (m/v) hydroxylamine hydrochloride, and after that, they were treated with 8 mmol/L KBr and 1.6 mmol/L KBrO3 in an hydrochloric medium. Atomic fluorescence spectrometry measurements were made by multicommutation, which provides a fast alternative in quality control analysis due to the easy treatment of a large number of samples (approximately 70/hr). It is an environmentally friendly procedure, which involves a waste generation of only 94.5 mL/hr as compared with the 605 mL/hr obtained by using continuous AFS measurements. The limit of detection found was 0.011 ng/g Hg in the original sample. The method provided a RSD of 3.4% for five independent analyses of a sample containing 0.30 ng/g Hg. Results obtained for commercially available milk samples varied between 0.09 and 0.61 ng/g Hg depending on the type of sample and its origin. The source of measurement uncertainty of trace mercury in vegetables by CV-AFS were analyzed and evaluated by Ma and Xiangli (2007). The main uncertainty sources were the preparation of standards, the weighing process of sample, the digestion of sample, the constant volume of sample, and repeatability of determination of results. When mercury content in vegetables was 5.2 μg/kg, the expanded uncertainty was 0.50 μg/kg.

References Alkemade C.T.J. and J.M.W. Milatz. A double-beam method of spectral selection with flames. Appl. Sci. Res. 1955, B4:289–299. Butcher D.J. and J. Sneddon. A Practical Guide to Graphite Furnace Atomic Absorption Spectrometry. John Wiley & Sons, New York, 1997.

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Cava-Montesinos P., E. Rodenas-Torralba, A. MoralesRubio, M. Luisa Cervera, and M. de la Guardia. Cold vapor atomic fluorescence determination of mercury in milk by slurry sampling using multicommutation. Anal Chimica Acta. 2004, 506(2):145–153. Dedina J. and D.L. Tsalev, eds. Hydride Generation Atomic Absorption Spectrometry. John Wiley & Sons, New York, 1995. Dyer C.A. Heavy metals as endocrine-disrupting chemicals. In: Endocrine-Disrupting Chemicals. A.C. Gore, ed. Humana Press, Totowa, New Jersey, 2007, pp. 111–134. Ferrieira H.L., S.L. Ferreira, M.L. Cerva, and M.L. de la Guardia. Development of a non-chromatographic method for the speciation analysis of inorganic antimony in mushroom samples by hydride generation atomic fluorescence spectrometry. Spectrochim Acta. 2009, 64B:597–600. Guldas M., A.F. Dagdelen, Adnan G. Biricik, and F. Gunnur. Determination and comparison of some trace elements in different chocolate types produced in Turkey. J Food Agr Environ. 2008, 6(3–4):90– 94. Haswell, S.J., ed. Atomic Absorption Spectrometry: Theory, Design and Applications. Elsevier Science Publishers, Amsterdam, 1991. Hontela A., A. Lacroix. Heavy metals. In: Endocrine Disruption. D.O. Norris and J.A. Carr, eds. Oxford University Press, Oxford, UK, 2006, pp. 356–374. Ingle J.D. and S.R. Crouch. Spectrochemical Analysis. Prentice Hall, Englewood, New Jersey, 1988. Ioannidou M.D., G.A. Zachariadis, A.N. Anthemidis and J.A. Stratis. Direct determination of toxic trace metals in honey and sugars using inductively coupled plasma atomic emission spectrometry. Talanta. 2005, 65(1):92–97. Jedrzejczak R. Determination of total mercury in foods of plant origin in Poland by cold vapour atomic absorption spectrometry. Food Addit Contam. 2002, 19(10):996–1002. Jorhem L., C. Aastrand, B. Sundstroem, M. Baxter, P. Stokes, J. Lewis, J. Peterssson, and K. Grawe. Elements in rice from the Swedish market: 1. Cadmium, lead, and arsenic (total and inorganic). Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2008, 25(3):284–292. Julshamn K., A. Magge, H.S. Norli, K.H. Groblecker, L. Jorhem, and P. Fecher. Determination of arsenic, cadmium, mercury, and lead by inductively coupled plasma/mass spectrometry in foods after pressure digestion: NMKL interlaboratory study. J AOAC International. 2007, 90(3):844–856. King A.S. [Discussion]. Astrophys J. 1905, 21: 236–243. Kingston H.M. and S.J. Haswell, eds. Microwaveenhanced Chemistry, Fundamentals, Sample Preparation, and Applications. American Chemical Society, Washingon DC, 1997. Lajunen L.H.J. Spectrochemical Analysis by Atomic Absorption and Emission. Royal Society of Chemistry, Cambridge, England, 1992. L’vov B.V. The analytical use of atomic absorption spectra. Spectrochim. Acta A. 1961, 17:761–770.

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Ma X., and X. Xiangli. Evaluation of measurement uncertainty of trace mercury in vegetables by coldvapour atomic fluorescence spectrometry. Huaxue Fenxi Jiliang. 2007, 16 (6):11–13. Massmann H. Vergleich von Atomabsorption und Atomfluores—zenz in der Graphitküvette. Spectrochim. Acta A. 1968, 23B:215–226. Milacic R., and B. Kralj. Determination of Zn, Cu, Cd, Pb, Ni, and Cr in some Slovenian foodstuffs. Eur Food Res Tech. 2008, 217(3):211–214. Montaser A. and D.W. Golightly, eds. Inductively Coupled Plasmas in Analytical Atomic Spectrometry, 2nd ed. VCH Publishers, New York, 1992. Nolte J. ICP-Emission Spectrometry-A Practical Guide. Wiley-VCH, Hoboken, New Jersey, 2003. Perello G., R. Marti-Cid, J.M. Llobet, and J.L. Domingo. Effects of various cooking processes on the concentrations of arsenic, cadmium, mercury, and lead in foods. J Agric Food Chem. 2008, 50(23):11262–11269. Rubio C., A.J. Gutierrez, C. Revert, J.I. Reguera, A. Burgos, and A. Hardisson. Daily dietary intake of iron, copper, zinc, and manganese in a Spanish population. Int J Food Sci Nutr. 2009, 60(7):590–600. Schoeters G., E. Den Hond, M. Zuurbier, R. Naginiene, P. Van Den Hazel, N. Stilianakis, R. Ronchetti, J.G. Koppe. Cadmium and children: Exposure and health effects. Acta Paediatr. 2008, 95(s453):50–54. Sneddon J. and C.A. Thibodeaux. Spectrochemical methods in the determination of metals in seafood. In: Handbook of Seafood and Seafood Products and Analysis, L.M. Nollet and F. Toldra, eds. 2009, CRC Press, Boca Raton, Florida, pp.751–771. Sneddon J., C. Hardaway, K.K. Bobbadi, and A.K. Reddy. Sample preparation of solid samples for metal determination by atomic spectroscopy: An overview and selected recent applications. Appl Spectros Rev. 2006, 41(1):23–42.

Sneddon J. Use of spectrochemical methods for the determination of metals in fish and other seafood in Louisiana. In: Determination of Chemical Elements in Food: Applications for Atomic and Mass Spectrometry, S. Caroli, ed. 2007, John Wiley & Sons, Hoboken, New Jersey, pp. 437–454. Sneddon, J. ed. Sample Introduction in Atomic Spectroscopy, Elsevier Science, Amsterdam, the Netherlands, 1990. Sneddon J., P.W. Rode, M.A. Hamilton, S. Pingeli, and J.P. Hagen. Determination of metals in seafood. Appl Spectros Rev. 2007, 42(1):1–16. Sobelman I.I. Atomic Spectra and Radiative Transitions, 2nd ed. Springer-Verlag, Berlin, 1992. Soceanu A., S. Dobrinas, V. Popescu, and V. Magearu. Determination of total chromium in some vegetables and fruits grown in urban and rural areas. Analele Universitatii “Ovidius” Constanta, Seria: Chimie. 2008, 19(1–2):27–29. Stoica A., B.S. Katzenellenbogen, M.B. Martin. Activation of estrogen receptor by the heavy metal cadmium. Mol Endocrinol. 2000, 14 (4):545– 553. Tinggi U., C. Reilly and C. Patterson. Determination of manganese and chromium in foods by atomic absorption spectrometry after wet digestion. Food Chem. 1997, 60(1):123–128. Walsh A. The application of atomic absorption spectra to chemical analysis. Spectrochim. Acta. 1955, 7:108–117. West T.S., and X.K. Williams. Atomic absorption and fluorescence spectroscopy with a carbon filament atom reservoir. I. Construction and operation of atom reservoir. Anal. Chim. Acta. 1969, 45:27–41. Woodriff, R. Atomization chambers for atomic absorption spectrochemical analysis: A review. Appl. Spectros. 1974, 28(5):413–417.

Chapter 13 Surfactants Bing Shao

Introduction Surfactants are chemicals that stabilize mixtures of oil and water by reducing the surface tension at the interface between the oil and water molecules. Surfactants have many different types. The most accepted and scientifically sound classification of surfactants is based on their dissociation in water. Based on the ionization characteristics in aqueous solution, they are divided into four main categories: anionic, cationic, zwitterionic, and nonionic. Anionic surfactants are the most commonly used surfactants, including alkylbenzene sulfonates, alkyl sulfonates, fatty acid salts, lauryl sulfate, perfluorinated carboxylates dialkyl sulfosuccinate, lignosulfonates and sulfonates, etc. Cationic surfactants are dissociated in water into an amphiphilic cation and an anion, most often of the halogen type. A very large proportion of this class corresponds to nitrogen compounds such as fatty amine salts and quaternary ammoniums, with one or several long chains of the alkyl type, often coming from natural fatty acids. Nonionic surfactants account for about 45% of industrial production. They do not ionize in aqueous solution because their hydrophilic group is of a nondissociable type, such as alcohol, phenol, ether, ester, or amide. They include ethoxylated linear alcohols, ethoxyl-

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

ated alkyl phenols, fatty acid esters, amine and amide derivatives, alkyl polyglucosides, propylene oxide copolymers, ethylene oxide, polyalcolols and ethoxylated polyalcohols, thiols (mercaptans) and derivates, etc. When a single surfactant molecule exhibits both anionic and cationic dissociations, it is called amphoteric or zwitterionic. This is the case for synthetic products such as betaines or sulfobetaines and natural substances such as amino acids and phospholipids. Among these surfactants, only alcaline (sodium, ammonium, potassium) salts, sulfo-carboxylic compounds, fatty acid esters, esters of hexitols, and cyclic anhydrohexitols such as TWEEN and SPAN brand, etc., can be used as food additives (Drew 2006). Perfluorinated carboxylates, sulfonates, and alkylphenol ethoxylates (APEOs) are prohibited for use in food industries due to their toxicity. Although aquatic toxicity data are widely available for anionic, cationic, and nonionic surfactants, APEOs and perfluorinated carboxylates and sulfonates are of the most concern in the last two decades due to their endocrine-disrupting properties (Knepper et al. 2003; Ying 2006). In this chapter, only APEOs and their metabolites are discussed. Perfluorinated carboxylates and sulfonates will be introduced in Chapter 16.

Alkylphenol ethoxylates and alkylphenols Alkylphenol ethoxylates (APEOs) are commonly used as nonionic tensides, dispersive agents in paper and leather manufacturing, 305

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APEOs R

O

( CH CH O (n CH CH OH 2

2

2

2

n=3~50

R

O

( CH CH O (mCH CH OH 2

2

2

R

2

O

( CH CH O (mCH COOH 2

2

m=2~(n–1)

APECs R

2

O

( CH CH O (i CH CH OH 2

2

2

R

2

O

( CH CH O (i CH COOH 2

2

2

i=0, 1

R

OH AP

Figure 13.1. Degradation pathway of APEOs.

emulsifiers for pesticide formulations, and as auxiliary agents for drilling and flotation. The most important members are nonylphenol ethoxylates (NPEOs) and octylphenol ethoxylates (OPEOs), which account for approximately 80% and 20% of the total APEO production. The number of ethoxylate units can be as high as 100, whereas the most commonly used technical products usually are mixtures with average ethoxylate numbers of 9 and 10. This kind of surfactant was produced in huge quantities; the annual worldwide usage was estimated to be around 650,000 tons. Alkylphenols (APs) are the raw materials for synthesis of APEOs as well as the main breakdown product of the latter. APEOs are degraded into corresponding short-chain ethoxylates and carboxylic acid derivatives (NPECs) (Figure 13.1) (Giger et al. 1984; Ahel et al. 1994a, b; Jonkers et al. 2001; Okayasu et al. 2005). Nonylphenol (NP) and octylphenol (OP) are regarded as more toxic than their parent compounds (Jobling et al. 1996).

Endocrine-disrupting activities of alkphenolic compounds A large number of the experiments in vivo or vitro confirmed that APs pose endocrinedisrupting activity causing various effects on the reproductive development of mammals and aquatic organisms. Dodds and Lawson reported the estrogenic properties of APs as early as 1938 (Dodds and Lawson 1938). Forty years later, Mueller and Kim (1978) found that 4APs can replace estradiol from the estrogen receptor. It has also been shown that nonylphenol may induce a response to estrogen-sensitive MCF7 human breast tumor cells. White et al. (1994) showed that some alkylphenols were estrogenic in fish, birds, and mammals. An advancement in vaginal opening had been found for NP-exposed prepubertal rats, along with disruptions in estrous cyclicity and alterations in pituitary hormone levels (Chapin et al. 1999; Laws et al. 2000; Kim et al. 2002). Several studies have demonstrated that NP affects teleost reproduction (Arukwe et al. 1997; Burkhardt-Holm et al.

Surfactants

2000; Hemmer et al. 2001). Notably, NP induces the female-specific, egg yolk precursor vitellogenin (VTG) in males (Matozzo and Marin 2005; Matozzo et al. 2008). Shortchain nonylphenol monoethoxylate (NPEO1) and nonylphenol diethoxylate (NPEO 2) and NPEC1 were proved to pose estrogenic potencies similar to NP (Jobling et al. 1996). A most recent study suggested that NP exposure could disturb pubertal development, and pubertal girls seem more sensitive than pubertal boys to the exposure of endocrine-disruption chemicals (EDCs) (Chen et al. 2009).

Occurrence of alkylphenolic compounds in food matrices Due to the endocrine-disrupting activities, APEOs and their metabolites have raised public concerns over their ecological effects not only in environmental science but also in food science, especially in food safety and quality monitoring. In the past decade, there has been growing attention to the presence of alkylphenolic compounds in foodstuffs, covering a wide range of physical types of matrix, from fish and animal tissues to milk and cereal. Fish and shellfish are the most common biosamples because they are typical aquatic organisms that might be sensitive to the APEOs and APs in surface water or wastewater. NP concentration levels have been investigated in mollusks such as clams, cuttlefish, squid, and mussels from marine ecosystems. Wenzel et al. (2004) reported the retrospective monitoring of NPs in common mussels (Mytilusedulis) from the North Sea and Baltic Sea. The study clarified the fact that NPs were detected in all mussel samples from 1985 to 1997, with the highest value of 9.7 ng/g wet wt. at Eckwarderhorne in 1985, whereas the 4-NP concentrations were lower than the limit of quantification (2 ng/g wet wt.) after 1997. The concentrations detected in clams and mussels from the Adriatic Sea were 243– 265 ng/g lipid (Ferrara et al. 2001). NP,

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NPEO1, and NPEO2 were detected in all shellfish samples obtained from rivers flowing into Lake Biwa, with concentration ranges of 2.8–19.3 ng/g, 7.7–23.3 ng/g, and 2.0–5.3 ng/g wet wt., respectively (Tsuda et al. 2002). Fish taken from the Chongqing area of China in December 2000 were found to contain 4-NP of about 1.9 μg/g and NPEOs of 0.4–48.3 μg/g, with the highest concentration level found in liver (Shao et al. 2005b). A concentration range of 151–300 ng/g lipid was observed in six fish species (e.g., weever, catfish, bartail flathead, white flower croaker, wolffish, and mullet) from Bohai Bay by Hu et al. (2005b), who also detected NPEOs in these fish samples with total concentration levels from 183.9 ng/g lipid to 678.2 ng/g lipid. Pojana et al. (2007) reported a survey on the occurrence and distribution of natural and synthetic EDCs in the Mediterranean mussel (Mytilus galloprovincialis) in the Venice lagoon, and NPs were detected at high frequency with a concentration range of 115–211 ng/g dry wt. The method was applied to the analysis of NPs in mussels from the Masan Bay of Korea, where concentrations ranged from 50.5 to 289.2 ng/g dry wt. (Wang et al. 2007). In 2002, Güenther et al. measured the levels of NPs in 39 foods and beverages from German supermarkets, including fruits and vegetables, dairy products, fish and meat, bread, pasta, beer, coffee, and chocolate, and found a concentration range of 0.1–19.4 μg/ kg (fresh weight). This is the first comprehensive analysis of NP in food and reveals that the chemicals are ubiquitous in German food products, including baby food. The findings suggest that food may be an important route of exposure to endocrine-disrupting compounds in humans (Erickson 2002; Güenther et al. 2002). In Taiwan, a similar study was performed by Lu and her colleagues (2007) by sampling 25 types of food typically consumed by locals, including two freshwater fish, two saltwater fish, two shellfish, two other seafoods, two poultry, two livestock, two

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vegetables, one rice and noodle, three other proteins, and three fruits. The foodstuff samples were analyzed for five alkylphenol compounds; NP, OP, butylphenol (BP), nonylphenol monoethoxylate (NPEO1), and nonylphenol diethoxylate (NPEO2). NP was detected most frequently (76.4%, 243/318) in the samples, with average concentrations of 235.8 ± 90.7 μg/kg wet wt. The second and third most frequently detected were BP and OP, respectively. Only salmon contained NPEO2 in an average concentration of 241.8 ± 252.2 μg/kg wet wt. Salmon and oysters contained NPEO1, averaging 9.8 ± 29.4 and 5.2 ± 4.7 μg/kg wet wt., respectively. Estimated daily intake of NP based on the Taiwanese survey was 31.40 μg/ day, which is 4-fold and 8.5-fold higher than the daily intake in Germany and New Zealand, respectively. Rice was the major source of NP intake in Taiwan. All these results indicate that APs (especially NP) are ubiquitous in food. A question is raised regarding the source of this chemical. It is easy to understand that NP in aquatic biota is due to water contamination, which has been proved by numerous publications (Staples et al. 1998; Snyder et al. 2001b; Tsuda et al. 2001; Wenzel et al. 2004; Lietti et al. 2007). According to Güenther et al. (2002), NPs in vegetables can likely be attributed to pesticides. NPEOs that degrade to NPs are commonly used as emulsifying agents in pesticide formulations. Another source might be plastic packaging materials from which NPs, used, for example, in tris(nonylphenol)phosphite as an antioxidant, could migrate into food. Several researchers have studied the migration of NP from plastic containers involving polyvinyl chloride (PVC) films, high-density polyethylene (HDPE), and ploy(ethylene terephthalate) (PET) into food stimulants and foods, such as rice, water, and milk, based on FDA guidelines for migration testing or microwave oven heating (Inoue et al. 2001; Loyo-Rosales et al. 2004). These tests confirmed that one of the origins of these compounds is the plastic

containers. The results have been summarized in a recently published paper (Muncke 2009).

Analysis of alkylphenolic compounds in foodstuffs Investigation of alkylphenolic compounds in food samples created a need for the development of improved multiresidue, multimatrix, and multitechnique analytical methods. In almost all cases, there is a sample preparation method prior to the determination by the standard analytical techniques (preconcentration, extraction, purification, derivation, etc.; Figure 13.2). The following section gives an overview of the analytical methods based on the available literature. The main experimental conditions used in the analyses are listed in Table 13.1.

Sample extraction Strategies for sample handing strongly depend on the nature of the analytes and matrices involved and on the concentration levels of the analytes. Among these three factors, the nature of the analyte (physical and chemical properties) is the first concern. Logarithmic value of the partition coefficient of one chemical in the octanol/water system (log Kow) is an indicator of the lipophilicity. A high log Kow is typical for hydrophobic compounds, whereas a low Kow signifies a compound easily soluble in water. Log Kow of OP, NP, and NPEO1–3 were between 3.9 and 5.6 (Ahel and Giger 1993a, b; Gundersen 2001), suggesting that these substances pose relatively high lipophilicity and may become associated with organic matter. Due to this property, organic solvents such as n-hexane, dichloromethane (DCM), ethyl ether, methanol, and acetonitrile are commonly used as extraction agents. For this step, ultrasonic extraction (Casajuana and Lacorte 2004; Pojana et al. 2007); accelerated solvent extraction (ASE) (Schmitz-Afonso et al. 2003; Gadzala-Kopciuch et al. 2008); Soxhlet

Surfactants

309

Food sample

Homogenization Surrogate standard Extraction ASE, MSPDE, Soxhlet extraction, steam distillation solvent extraction, SPE

Purification GPC, column chromatography, SPE, HPLC fractionation Internal standard Derivatization Methylation, silylation, pentafluorobenzylation

LC, LC-MS, LC-MS/MS

GC-MS Figure 13.2. Schematic diagram of the analytical method of alkylphenolic compounds in foodstuffs. (The dotted box and arrow indicate an optional step in the process.)

extraction (Shao et al. 2005b; Wang et al. 2007); steam distillation solvent extraction (Gunther et al. 2001; Snyder et al. 2001a; Kannan et al. 2003); and matrix solid-phase dispersion extraction (MSPDE) (Zhao et al. 1999; Shao et al. 2007b) are frequently applied in food matrix analysis. Danzo et al. (2002) used a simple ethyl acetate extraction with NaCl added in a vortex tube to determine NP concentrations in serum of exposed guinea pigs using GC/MS. Performed by Meier et al. (2005), APs involving OP and NP were extracted by DCM, with anhydrous sodium sulfate added, to remove the water in cod tissues. It is reported that ultrasonic-assisted extraction could improve the extraction efficiencies of analytes from the matrix. The ability of ultrasonic treatment to enhance the recovery of chemicals is mainly attributed to

its facilitation of mass transfer between immiscible phases through agitation via microjetting and microstreaming, especially at low frequency (Vinatoru et al. 1997). In a paper by Pojana et al. (2007), NP, NPEC1, and other EDCs were sonicated with a nhexane/acetone mixture (70 : 30, v : v) for 2 h (20 mL, two times) from 5 g of freeze-dried mussel soft tissue. Commercial whole milk samples, including ultrahigh temperaturetreated (UHT-treated) milk, in bottled sterilized milk and powdered milk infant formula, were treated by methanol and followed by sonication for NP determination (Casajuana and Lacorte 2004). ASE, also called pressurized liquid extraction (PLE), is a fully automated technique that uses common solvents to rapidly extract solid and semisolid samples at elevated temperatures and pressures. It was widely used in

310 Sample Fish and shellfish Rainbow trout muscle and zebra mussels Fish and shellfish Blue mussels

Fish

Fish tissue 39 foodstuffs, including peanut cream, gooseberry, marmalade, beer, infant food, coffee, sugar, and so on

Compound

NP and OP

OP and NPEOs

NP, NPEO1-2, OP, BPA, BP

NP

NP, NPEO1-3

NP, NPEO1-5

NP

Steam distillation solvent extraction Cleanup by HPLC fractionation (Hypersil APS column, 125 mm × 4 mm, 3 μm) Derivated using MTBSTFA 4-n-NP as a surrogate

Steam distillation solvent extraction Cleanup by HPLC fractionation (Hypersil APS column, 125 mm × 4 mm, 3 μm) 4-n-NP as a surrogate Steam distillation solvent extraction Cleanup by HPLC fractionation (Luna silica column, 250 mm × 4.6 mm, 5 μm) Cumylphenol as a surrogate ASE with DCM Purification by a NH2 SPE cartridge

Liquid–liquid extraction Purification by a Florisil column

MSPDE using C18 powder and sequential Al2O3 cleanup

Liquid–liquid extraction Purification by a Florisil column

Pretreatment Procedure

HPLC-FL Hypersil APS column, 100 mm × 4.6 mm, 5 μm Large-volume-injection GC/MS CPSil8 CB column, 30 m × 0.25 mm, 0.25 μm

GC-MS DB-17 MS column, 30 m × 0.25 mm, 0.25 μm

GC–MS DB-5 MS column, 60 m × 0.25 mm, 0.25 μm

GC-MS DB-1701 column, 30 m × 0.53 mm, 1.0 μm HPUV-for screening and HPLC-FL for quantitation Lichrospher 100RP-18 column, 125 × 4 mm, 5 μm HPLC-FL Inertsil PH column, 150 × 4.6

Detection

Table 13.1. Main experimental conditions used in the analysis of alkylphenolic compounds in food samples. Limit of detection

27 ng/g

5–44 ng/g

3.3, 16.8, 18.2, and 20.6 ng/g for NP, NPEO1, NPEO2, and NPEO3, respectively

2 ng/g for NP, NPEO1-2 and 1 ng/g for other 3 compounds The detection limit was 15 ng NP absolute

10 ng/g for OP and 30 ng/g for NPEOs

20 ng/g for NP and 2 ng/g for OP

Reference

Güenther et al. 2002

Datta et al. 2002

Keith et al. 2001; Snyder et al. 2001a; Kannan et al. 2003

Güenther et al. 2001

Tsuda et al. 2000a

Zhao et al. 1999

Tsuda et al. 1999, 2000b

311

Osprey eggs, carp, lake trout and walleye

Commercial whole milk

Animal feed Cockles, mussels, oysters, and scallops Norway lobster, spottail mantis shrimp, and six fish species Cod muscle and liver

Biota samples from river and marine

NP, OP, NPEO1-5, and OPEO1-5

NP, BPA, and other EDC compounds

OP and NP

NP

NP, OP, OPEOs, and NPEO1-4

Alkphenols from phenol to nonylphenol

NP and NPEO1-25

Sample Human breast milk

NP, BPA

Compound

Pretreatment Procedure

Extraction with DCM Cleaned up by GPC Derivatized with pentafluorobenzoyl chloride 2 H-labeled chemicals as surrogate standard Soxhlet extraction with methanol/ DCM (3 : 7) Purification by NH2 or GCB SPE cartridge

Extraction by methanol Cleanup by C18 SPE cartridges and Florisil cartridges 4-n-NP as a surrogate Stir bar sorptive extraction and online SPE purification by a TSK BSA-ODS/S precolumn Extraction with hexane followed by cleanup on NH2 SPE cartridge Liquid-liquid extraction

Liquid–liquid extraction followed by cleaning with NH2 SPE cartridge Derivatization by BSTFA NP-d4 and BPA-d16 used as surrogate standards ASE with DCM SPE cleanup by C18, SPE cleanup by NH2 cartridge (a further SPE cleanup by C18 for LC-MS/MS analysis)

Detection

GC-MS for NP, NPEO1-2 HP-5 MS column, 30 m × 0.25 mm, 0.25 μm LC-ESI-MS for NPEO3-25 a Capcell Pak C18 precolumn (50 mm × 2.1 mm, 3 μm) and a Spherisorb SW3 silica analytical column (150 mm × 2.1 mm, 3 μm)

GC-MS Rtx-5 column, 15 m × 0.25 mm Dodecylbenzene as an internal standard GC-MS DB-5 MS column, 60 m × 0.25 mm, 0.25 μm

LC-MS Mightysil RP-18 GP column (100 mm × 2.0 mm, 5 μm) Biacore biosensor immunoassay

LC-ESI-MS/MS (MSpak GF-310 4D column, 150 mm × 4.6 mm, 5 μm) compared with HPLC-FL (Hypersil APS column, 100 mm × 4.6 mm, 5 μm) 13 C-labeled chemicals as internal standards for LC-MS/MS analysis GC-MS HP-5 MS column, 30 m × 0.25 mm, 0.25 μm

GC-MS Phenanthrene-d10 as a internal standard

Limit of detection

Reference

5.0, 20.0, 30.0, 5.0, and 2.0 ng/g (wet wt.) for 4-NP, NPEO1, NPEO2, NPEO3, and NPEOs (n>3), respectively.

(continued)

Hu et al. 2005b; Shao et al. 2005b

Meier et al. 2005

Ferrara et al. 2001, 2005

0.5 μg/l, 8.1 μg/l, 1.1 μg/l for OP, NP, and OPEOs, respectively 6.35–22.72 ng/g

Samsonova et al. 2004

Kawaguchi et al. 2004

Casajuana and Lacorte 2004

Schmitz-Afonso et al. 2003

Otaka et al. 2003

10 ng/g

1 ng/g for OP and 5 ng/g for NP

0.18 ng/g for NP

LC-MS/MS: 4–12 ng/g in eggs, and 6–22 ng/g in fish

0.09 ng/g and 0.50 ng/g for BPA and NP, respectively

312 Mussels

Pork, fish, rabbit, duck meat, and chicken Eggs and milk

Mussels and oysters

NP, OP, and BPA

NP, OP, and BPA

Alkphenols including NP and OP

Direct immersion SPME and headspace on-fiber silylation by BSTFA Sonicated extraction with an n-hexane/acetone mixture (70 : 30, v : v) Further purified with 2 g Florisil. ASE with acetone, purification by a NH2 SPE cartridge 4-n-NP as a surrogate MSPDE using C18 as dispersant Purification by a NH2 SPE cartridge 4-n-NP as a surrogate

Fish blood

NP, NPEC1, and other EDCs

ASE with methanol purification by an Oasis HLB SPE cartridge

Corn breakfast cereals

NP, BPA, and five other phenolic EDCs NP, OP, and other EDCs

Soxhlet extraction and purification by a tandem Florisil cartridge Derivation by BSTFA during purification

Steam distillation solvent extraction

Fresh fruits and vegetables

OP, NP, OPEO1, and NPEO1

Pretreatment Procedure SPE using an Oasis HLB cartridge 4-n-NP as a surrogate

Sample Mineral drinking water and soda beverages

Compound NP, OP, and BPA

GC-MS DB-5 MS column, 30 m × 0.32 mm, 0.25 μm Phenanthrene-d10 as an internal standard

LC–ESI–MS/MS Symmetry C18 column, 150 mm × 2.1 mm, 3.5 μm LC–ESI–MS/MS Symmetry C18 column, 150 mm × 2.1 mm, 3.5 μm

GC-MS DB-5 MS column, 30 m × 0.25 mm, 0.25 μm [2H14]-p-terphenyl as an internal standard LC-ESI-MS XTerra MS C18 column, 2.1 × 100 mm, 3.5 μm GC-MS HP-5 MS column, 30 m × 0.25 mm, 0.25 μm HPLC–ESI-MS

Detection LC–ESI–MS/MS Symmetry C18 column, 150 mm × 2.1 mm, 3.5 μm

1.0, 0.2, and 0.4 ng/g for BPA, NP, and OP, respectively BPA, NP, and OP: 0.10, 0.10, and 0.25 ng/g for eggs; 0.10, 0.05, and 0.10 ng/g for milk, respectively. 3 ng/g for NP and OP 1 ng/kg for other APs

0.2–5.0 ng/g dry wt.

0.043 μg/g for BPA and 0.003–0.013 μg/g for other analytes 0.01 μg/L for OP and 0.2 μg/l for NP

Limit of detection BPA, NP and OP: 0.04, 0.03, and 0.2 ng/L for 2 L of mineral drinking water and 2.0, 1.8, and 8.0 ng/L for 50 mL of soda beverages, respectively Less than 0.2 ng/g in 10 g (fresh weight) of sample

Table 13.1. Main experimental conditions used in the analysis of alkylphenolic compounds in food samples. (cont.)

Wang et al. 2007

Shao et al. 2007b

Shao et al. 2007a

Pojana et al. 2004, 2007

Yang et al. 2006

Carabias-Martinez et al. 2006

Yang and Ding 2005

Reference Shao et al. 2005a

313

ASE with hexane or actonitrile Purification by a NH2 SPE cartridge Liquid–liquid extraction

Fish tissues Human breast milk

Breast milk and commercial cow milk products

4-n-NP and 4-tert-OP

NP, OP, NPEO1-2, and OPEO1-2

OP, NP

Extraction with n-hexane and dilution with 50% methanolic-water Purification by an Oasis HLB SPE cartridge Cumylphenol as a surrogate

Steam distillation extraction with n-hexane without purification Liquid–liquid extraction Derivatized with trifluoroacetic anhydride

OP, NP

One spot tail mantis shrimp, one common octopus, and nine fish species

Pretreatment Procedure Extraction by acetonitrile and purification by a florisil column

NP, OP, BP, and NPEO1-6

Sample 25 types of food typically consumed in Taiwan, including meat, fish, vegetables, rice, noodles, fruits, and so on Seven fruit purees and three meat puree samples

NP, OP, BP, and NPEO1-2

Compound

Detection

GC-MS DB-XLB column 30 m × 0.25 mm, 0.25 μm Dodecylbenzene (DB) used as an internal standard GC-MS DB-5MS column, 15 m × 0.25 mm, 0.1 μm

GC-MS DB-5MS column, 15 m × 0.25 mm, 0.1 μm Cumylphenol as an internal standard GC-MS DB-XLB column, 30 m × 0.25 mm, 0.25 μm Dodecylbenzene as an internal standard HPLC-UV Supelcosil LC-C18-DB column

HPLC-FL Luna C18-A column, 150 mm × 4.6 mm, 5 μm, with a C18 guard column, 4 mm × 3 mm

less than 0.05 ng/g from 20 g samples

Lin et al. 2009

Ademollo et al. 2008

Gadzala-Kopciuch et al. 2008

0.18 and 0.16 ng/μL for NP and OP, respectively 0.023–86.9 ng/mL

Ferrara et al. 2008

Li et al. 2008

0.5–44.3 ng/g

0.2 ng/g wet wt.

Reference Mao et al. 2006; Lu et al. 2007

Limit of detection 5.4 ng/g for OP, 5.2 ng/g for BP, 8.9 ng/g for NP, 8.7 ng/g for NPEO2, and 8.1 ng/g for NP1EO

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Analysis of Endocrine Disrupting Compounds in Food

the analysis of trace contaminants in foodstuffs (Abend et al. 2003; Klejdus et al. 2004; Urraca et al. 2004). Datta et al. (2002) used this technique to extract ground fish tissue for quantification of NP and its ethoxylates, up to NPEO5. Shao et al. (2007a) applied ASE for determination of NP, OP, and bisphenol A (BPA) in meat by HPLC and a variety of extraction solvents (e.g., dichloromethane, acetone, methanol dichloromethane). Other parameters and purification conditions, were also detailed in the study. Schmitz-Afonso et al. (2003) used ASE to establish the analytical method of NP, OP, and their ethoxylated compounds in osprey eggs and fish. The homogenized samples were extracted using DCM and then further purified by solid-phase extraction (SPE). Rhind et al. (2005) applied DCM-heated extraction and SPE purification for analysis of APs from sheep liver, kidney, and muscle tissue. A sensitive method based on ASE and liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) has been developed for the determination in cereal samples of NP and six other EDCs, such as BPA, 4-tert-BP, etc. (Carabias-Martinez et al. 2006). Methanol was selected as the extraction solvent for ASE in this study. Soxhlet extraction, invented in 1879 by F. von Soxhlet and originally designed for the extraction of lipid from a solid material, is another effective technique for organic chemical extraction from solid samples. McLeese et al. (1981) studied the accumulation of AP in aquatic fauna, including salmon, with fish samples Soxhlet extracted using ethyl acetate. Concentrations of the weak EDCs (i.e., NP, OP, and NPEO1-2) were measured in a series of samples of tissues of wild fish from the River Aire and from a laboratory dosing experiment (Blackburn et al. 1999). The samples were extracted with DCM in a Soxhlet extractor. Hu and her colleagues developed a Soxhlet extraction procedure for EDCs (including NP, NPEOs, polycyclic aromatic hydrocarbons [PAHs], and pesticides)

in a wide range of biosamples, such as plankton, invertebrates, fish, and marine birds. DCM/methanol (70 : 30) was used as the extraction solvent to attain the desired compound (Hu et al. 2005b; Shao et al. 2005b). The method was also suitable for sediment and sludge (Hu et al. 2005a; Jin et al. 2007). Wang et al. (2007) presented a method for AP detection from mussels and oysters using Soxhlet extraction. A more recent paper reported that more than 100 oyster samples were treated by this technique to detect NPs and study its bioaccumulation (Vincent and Sneddon 2009). The Soxhlet extraction process is always repeated for 16 to 24 h to ascertain the high extraction rate. Steam distillation is one of the oldest methods of separating chemicals on the basis of differences in vapor pressures over water. Conventionally, this technique uses an external steam generator that jets steam into the sample and collects the chemicals by condensing the steam and volatile chemicals in a cooled flask. The steam distillation apparatus designed by Veith and Kiwus (1977) modified the Nielsen-Kryger apparatus (Nielsen and Kryger 1969) and could provide exhaustive distillation of pesticides and industrial chemicals from matrices. Güenther et al. (2001) reported an ultra-trace analysis method of NP in mussels from the German North Sea based on steam distillation and solvent extraction. Subsequently, they applied this process to extract NP in 60 different foodstuffs commercially available in Germany (Güenther et al. 2002). The solvent for the extraction was obtained by dissolving 40 g of NaCl and 2 mL of hydrochloric acid in 600 mL of water. The solvent was filled in the above-mentioned apparatus and purified by boiling and simultaneous extraction of the condensate with 20 mL of cyclohexane/iso-octane (1 : 1, v/v) for 5 h. After replacement of the aqueous and overlying organic phase in the steam distillation apparatus with fresh solvents, 15–80 g of food sample was placed in the distilling flask and suspended in the extraction solvent. The

Surfactants

samples were distillated for 5 h by simultaneous extraction with 20 mL of cyclohexane/ iso-octane (1 : 1, v/v). Keith et al. (2001) and Snyder et al. (2001a) studied the occurrence of NP and NPEO1-3 in fish tissues by extracting target compounds via this process. A simple and sensitive method for determining the alkylphenolic compounds (OP, NP, OPEO1, and NPEO1) in fresh fruits and vegetables involving extraction of a sample by a modified Nielson–Kryger steam distillation extraction using n-hexane for 1 h and GC-MS detection was conducted by Yang and Ding (2005). MSPDE, initially introduced by Barker et al. in 1989, is a sample preparation technique for solid and semisolid samples using bonded-phase solid supports as an abrasive to produce disruption of sample architecture and a bound solvent to complete sample disruption during the sample blending process. MSPDE techniques have been successfully applied to isolate a wide range of compounds, such as antibiotics, pesticides, and betaagonists from a variety of complex plant and animal samples (Long et al. 1990; Barker et al. 1993; Crouch and Barker 1997; Barker 2007). Application of MSPDE in food analysis can greatly reduce analysis time, the volume of solvent, and the expense of the purchase and disposal. A wide variety of solid extraction materials can be used, but for the nonionic surfactants usually a reversed-phase C18 type of sorbent is applied. Zhao et al. (1999) developed a modified MSPDE method with sequential cleanup over alumina to isolate and purify OP, NP, and NPEOs in fish and mussels. Between 0.5 and 1 g of biological tissue was weighed and transferred into a coated mortar. Two to 3 g of prewashed C18 powder was added, and the mixture was ground with a coated pestle until a freeflowing powder was obtained. A 20-mL glass syringe with a glass plunger was used to construct the MSPDE column. The process and sequential cleanup adsorbents were packed into the syringe barrel, where the extraction

315

and purification were carried out. Three grams of deactivated aluminum oxide were transferred into the barrel, and another filter was put on top of the Al2O3. A 0.2 μm filter unit was attached to the tip of the syringe to filter all the eluate from the column. The Al2O3 and filters were washed with 10 mL of methanol before the MSPD elution, and the collected washout was discarded. The mixture powder of C18 tissue was then transferred onto the column, and the syringe was tapped slightly to remove the air pockets inside the material. The method was applied by Uguz et al. (2004) in NP extractions and measurements from the liver of rainbow trout (Onchorynchus mykiss). Shao et al. (2007b) established a MSPDE method using C18 as dispersant for the extraction of NP, OP, and BPA in milk and egg samples. A subsequent cleanup step with amino-propyl SPE cartridges and LC/ESI/MS/MS was applied. Average recoveries of the whole method varied from 79% of BPA to 98% of NP, and relative standard deviations were equal to or lower than 15% for egg samples. The average recoveries in milk ranged from 86% to 84% for BPA, 90% to 99% for NP, and 82% to 103% for OP, and relative standard deviations were equal to or lower than 8%. SPE is an increasingly useful sample preparation technique. It can be directly applied to liquid samples including mineral water, purified water, and soft drinks. Reversedphase materials (e.g., C18, HLB cartridges) and graphitized carbon black cartridge (GCB) are the commonly used sorbents. A comprehensive analytical method by Shao et al. (2005a) based on LC/ESI/MS/MS with negative ionization mode has been developed for measuring OP, NP, and BPA in beverage samples. The concentration and cleanup were performed on a 200-mg Oasis HLB cartridge. This approach achieved satisfactory recoveries and low detection limits. Although solid-phase microextraction (SPME) is very simple, efficient, and widely used in environmental aqueous matrices, its

316

Analysis of Endocrine Disrupting Compounds in Food

application in biosamples for alkylphenolic compounds is very rare (Cai et al. 2004; Liu et al. 2008; Pan and Tsai 2008). Yang et al. (2006) established a fully automated method using a direct immersion SPME method with polyacrylate fiber for EDC extraction in fish blood. The extraction and derivatization time, ion strength, pH, incubation temperature, sample volume, and extraction solvent were optimized.

Purification In organic trace analysis, it is very important to obtain clean extracts prior to the chromatographic analysis in order to meet the low limits established by legislation, as well as to protect the chromatographic system. Thus the purification step must be carefully studied. Due to the complexity of food samples, further purification of the aqueous extracts is usually necessary to reduce the matrix interference. For this aim, several techniques have been developed and optimized, such as gel permeation chromatography (GPC) (McLeese et al. 1981), column chromatography (Blackburn et al. 1999), SPE (Shao et al. 2005b; Wang et al. 2007), and HPLC fractionation (Keith et al. 2001), with SPE being the most frequent. GPC is based on the ability of molecules to move through a column of gel that has pores of clearly defined sizes. The larger molecules cannot enter the pores, thus they pass quickly through the column and elute first. Slightly smaller molecules can enter some pores and thus take longer to elute, and small molecules can be delayed even further. The great advantage of the technique is that it is simple, it is isocratic, and large molecules rapidly elute. The technique has been used comprehensively in food analysis to separate target analytes from the lipid-rich extracts. McLeese et al. (1981) reported that fish samples were Soxhlet extracted and the lipids were removed by GPC on Biobeads using cyclohexane/ dichloromethane (1 : 1, v/v). DCM extracts were purified using a two-column GPC

coupled with a fluorescence (FL) detector to remove the large molecules, such as lipid for AP determination in cod tissues (Meier et al. 2005). The authors have instead coupled the two columns using an automatic injector as a switch vent. This design permits separation and discarding of most of the lipids before the sample enters into the second column, and thus there is a much better cleanup of the samples on the second column. Column chromatography is frequently used by organic chemists to purify liquids and solids. An impure sample is loaded onto a column of adsorbent, such as silica gel or alumina. An organic solvent or a mixture of solvents (the eluent) flows down through the column. Components of the sample separate from each other by partitioning between the stationary packing material (silica or alumina) and the mobile eluent. Molecules with different polarity partition to different extents and therefore move through the column at different rates. The eluent is collected in fractions. For APs and their related chemicals, normal phase sorbents, for example, Florisil and alumina, are the most commonly used stationary phase. As reported by Tsuda et al. (2000a, b, 2001, 2002), NP and OP in fish and shellfish are extracted with acetonitrile, and the lipids in the sample extract are eliminated by partitioning between hexane and acetonitrile. A cleanup procedure using a Florisil PR column followed. Investigations of NPs in food by Güenther et al. (2002) and Lu et al. (2007) were conducted by analysis of corresponding chemicals in a variety of foodstuffs. During the test, purification through Florisil column chromatography was used after sample extraction and concentration. This process by Blackburn et al. (1999), with alumina as the sorbent material, was carried out for the cleanup of marine fish samples. As mentioned above, SPE is an effective sample pretreatment technique that could be applied not only in sample extraction but also in sample purification. Conventional normalphase SPE cartridges, for example, Florisil

Surfactants

and NH2 cartridges, are the most commonly used sorbents for the extract purification during the determination of APs in food. Alternatively, reversed-phase SPE cartridges, e.g., C18 and Oasis HLB cartridges, are also applied in this field. Florisil is a highly selective adsorbent that is extensively employed in preparative and analytical chromatography. Recently, a cleanup kit composed of two Florisil columns and one adaptor was designed by Wang et al. (2007) for simultaneous measurement of several APs in mussels and oysters. Schematic diagram of cleanup unit were as follows. Column 1 contained 2 g of 5% deactivated Florisil absorbent. The nonpolar and slightly polar solvents were used as mobile phase. As a result, APs (in the slightly polar fraction) were separated from nonpolar lipids (in the nonpolar fraction) and highly polar lipids were retained in the column. Column 2 contained 1 g of fully activated Florisil absorbent. Only nonpolar solvent was used as mobile phase, so only silyl-derivatized APs (nonpolar) were eluted from the column and middle polar lipids were still retained in the column. The proposed method is a time-saving and economic technique with high selectivity and sensitivity. NH2 sorbent can use both hydrogen bonding and anion exchange. The mechanism of sorption is though hydrogen bonding from the hydroxyl group, etc., of the adsorbate to the amine group of the sorbent. It can be used as a polar sorbent, like silica, with different selectivity for acidic and basic analytes or as a weak anion exchanger in aqueous medium. For 4-NP, NPEO1, and NPEO2 analysis in the marine biosamples, Hu et al. (2005b) performed their sample purification through the means of an NH2 cartridge, which was conditioned with 9 mL acetone, 3 mL DCM, and 9 mL hexane, and then loaded with the extract of the biota samples, eluting with 8 mL hexane/2-propanol (90 : 10 v/v). In the same paper, the GCB cartridge was selected as the cleanup apparatus for NPEO (with EO unit from 3 to 25) detection. As for the reversed-

317

phase SPE cartridge, it should be noted that extracts diluting with water, or other aqueous solutions, were needed before sample loading to improve the retention and selectivity of APs upon SPE materials. For this reason, application of this kind of SPE cartridge might be limited in the field. Like column chromatography and SPE, HPLC fractionation is also used as a cleanup procedure in view of the different chromatographic behaviors between different compounds. Compared with column chromatography and SPE, HPLC allows a higher resolution, reproducibility, and mechanization. HPLC-FL was applied to remove lipid and fractionation collections by Güenther et al. (2002) and Keith et al. (2001). In these papers, the normalphase HPLC column, NH2 column, and silica column were used respectively. Note that background contamination and detection of analytes in procedural blanks are recognized problems (Kuch and Ballschmiter 2001; Snyder et al. 2001a; Rudel et al. 2003). As a matter of fact, some APs are commonly present in most indoor environments and it is, therefore, almost impossible to avoid some degree of detection of the analytes in procedural blanks (Soto et al. 1991; Rudel et al. 2003). Apparatuses made from rubber and plastic materials are regarded as main sources of NP. These should be avoided in the whole analytical procedure as much as possible. Alternatively, glass containers and vials, as well as polytetrafluoroethylene (PTEE) tubes and septums, are recommended. Before the analysis, all glassware was cleaned and then baked in a muffle furnace to remove the residue of organic chemicals. In addition, procedural blanks should be conducted for each batch of samples to understand the background contamination.

Detection Several methods have been proposed for the determination of APs and their relevant chemicals using gas chromatography mass

318

Analysis of Endocrine Disrupting Compounds in Food

spectrometry (GC/MS), high performance liquid chromatography (HPLC), and HPLC/ MS being the most favored. Gas chromatography–mass spectrometry Gas chromatography is regarded as the most powerful and versatile analytical separation method for organic compound determination, especially for volatile compounds. Detectors commonly used include the electron capture detector (ECD), flame ionization detector (FID), nitrogen phosphorus detector (NPD), mass selective detector (MSD), and so on. Although GC coupled to ECD and FID in AP/ APEO detection is reported, its application to food samples is rare. GC/MS is the predominant method for determination of alkylphenolic compounds in edible matrices because of its sensitivity and selectivity. Regarding the biosamples and foods, APEO analysis by GC/MS has been mainly used for more volatile biodegradation products of NP, sometimes accompanying OP and short-chain APEOs. Technical NP is a mixture of many isomers with different structures. Thus, GC/MS chromatograms of NP usually show several tens of peaks, whereas those of commercial OP are much less complicated (De Voogt et al. 1997; Thiele et al. 1997; Gundersen 2001). GC/MS, with, or without derivation, is frequently used for AP and relevant chemical determination in food. As for the former, selected ions at m/z 121, 135, and 149 for NP; as well as m/z 107 and 135 for OP, are the most commonly monitored. Ferrara et al. (2001) reported the first group of results on AP contamination of seafood in the Adriatic Sea. In their paper, they applied GC/MS with a 15mRtx-5 column to analyze alkylphenolic compounds in mollusks, including NP, OP, OPEOs, and NPEO1-4, which is APEO with the longest EO chains determined by this approach. Derivatization of APEOs has been carried out with a large number of derivation regents,

such as silylation derivatives N,O-Bis (trimethylsilyl)trifluoroacetamide (BSTFA) or N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and benzylation derivatives (pentafluorobenzoyl bromide or pentafluorobenzyl chloride). In addition to increased suitability and response, derivatization can improve resolution between coeluting compounds and overlapping peaks. More recently, Ferrara et al. (2008) investigated the occurrence of OP, NP, and their respective ethoxylates (with 1–6 ethoxylic group) in aquatic species of commercial interest from the Tyrrhenian Sea. Sample extracts were derivatized in a reaction vial by adding trifluoroacetic anhydride before injection in GC/MS with a DB-XLB 30-m column. Overall, GC/MS is the method of choice for the analysis of food samples containing APEO oligomers with six or fewer EO units. The low volatility, high polarity, and high molecular weight of related compounds with more EO units prevented their elution from the GC column. Although GC/MS presented a high resolution of NPs, few reports quantified individual amounts of the different isomers. During food analysis, most studies calculated the concentrations of NP by regarding the total area of detected peaks (Güenther et al. 2001, 2002). Comprehensive two-dimensional GC/MS was applied for analysis of technical NP, and resolution of 102 NP isomers was achieved (Ieda et al. 2005; Moeder et al. 2006). Recently, a GC/MS method has been developed by Zhang et al. (2007) for the simultaneous detection of three chiral NP isomers in water samples. However, these approaches have not been used in food sample determination. Previous studies have shown that the estrogenic effects and bioavailability of the individual NP isomers are heavily dependent on the structure of the side alkyl chain (Kim et al. 2004; Gabriel et al. 2005a, b). Occurrence of the NP isomer in food matrices should be considered in future studies.

Surfactants

Liquid chromatography APEO surfactants are complex mixtures consisting of various homologs and oligomers by length of the alkyl and ethoxylate chains. Polar normal-phase HPLC columns separate nonionic surfactants by their interaction with the hydrophilic EO chain without resolving the hydrophobes, whereas nonpolar reversedphase columns separate them by their interaction with the hydrophobic chain, only eluting the ethoxymers as a single peak (Thiele et al. 1997). Ultraviolet (UV) or FL detectors are the most commonly used because the aromatic ring chromophore in APEO molecules is sensitive to these types of detection. Generally speaking, FL detectors resulted in a 1 or 2 order of magnitude higher sensitivity than UV detectors. Thus, LC/UV is rarely applied in food analysis due to the high detection limit of the instrument and the trace level of alkylphenol in the matrix. Both normal-phase and reversed-phase LC/FL are frequently introduced in AP/APEO detection in variety of matrices. Its use in environmental samples has been summarized by Thiele et al. (1997). As for foodstuffs, determination of NP and NPEO1-5 in fish tissue using HPLC/FL on a normal-phase NH2 column is achieved with spectrofluorimetric detection at 275/300 nm, resulting in the method’s detection limits ranging from 5 ng/g to 44 ng/g (Datta et al. 2002). In another paper, a simple and highly sensitive method was described for the LC/FL determination of NP and NPEO1-2 in fish and shellfish, together with OP, BPA, and BP (Tsuda et al. 2000a). Analytes were separated on a reversed-phase column Inertsil PH and the excitation and emission wavelengths were set at 230 nm and 300 nm, respectively.

Liquid chromatography/mass spectometry LC, coupled to MS or tandem MS, has gained in popularity for contamination and residue analysis due to its high selectivity, specificity

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and sensitivity. It has been recognized as a robust analytical technique in food chemistry. LC/MS analysis of alkylphenol congeners has been attempted using both normal-phase and reversed-phase systems. Compared to atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI) usually offers a better specificity and sensitivity for APEOs/AP. Generally speaking, APEOs are analyzed under positive mode (ESI+) and NP, OP, and the carboxylated bioproducts are acquired under negative ESI mode, which leads to the production of [M-H]− ions. APEOs tend to form distinct adducts with ions originating from the mobile phase, the sample, or the introduction system (e.g., H+, Na+, K+, NH +4 ) and are sometimes doubly charged, resulting in different m/z. This has been documented in detail by Jonkers et al. (2005). LC/MS has been comprehensively applied in the identification and quantification of APEO/AP in foodstuffs. Shao et al. (2002) established an analytical method based on normal-phase LC/ESI/MS for determination of NPEOs in aquatic samples. Complete separation between each individual NPEO (with EO unit from 2 to 25) was achieved by combining a C18 precolumn with a silica analytical column and using acetonitrile–water as eluent. Consequently, this approach was used for the analysis of NPEOs in biota samples (Hu et al. 2005b; Shao et al. 2005b). SchmitzAfonso et al. (2003) compared the determination method of NP, OP, and their ethoxylated compounds (with EO1-5) in fish and osprey eggs by LC/FL and by LC/MS/MS. A normalphase aminopropyl silica column was equipped for LC/FL and a reversed-phase column was applied in the latter. As a result, LC/MS/MS provided the high sensitivity and specificity required for these complex matrices and an accurate quantitation with the use of 13C-labeled internal standards. Quantitation limits by LC/MS/MS ranged from 4 to 12 ng/g in eggs and from 6 to 22 ng/g in fish samples. Migration of nonylphenol from

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plastic containers to water and milk was studied using LC/MS/MS by Loyo Rosales et al. (2004). During LC/MS analysis, the electrospray response of the analytes in the complex matrix can be affected by nonanalyte-related coeluting components present in the original sample, even though these components themselves do not possess an electrospray response. This effect usually results in the signal suppression or less frequently in the enhancement of the analyte response (Benijts et al. 2004; Kloepfer et al. 2005; Villagrasa et al. 2007); it is highly related to the sample preparation procedure and determining condition. Matrix effect is one of the most important concerns when developing a LC/MS method. AP/APEO determination in food samples has been discussed in the literature (SchmitzAfonso et al. 2003; Carabias-Martinez et al. 2006; Shao et al. 2007a). The more accurate results in GC and LC are probably obtained using an internal standard. However, it can be difficult to find an appropriate substance that will elute in a position on the chromatogram that does not interfere or merge with any of the natural components of the sample extracts. Isotopelabeled chemicals and 4-n-NP are the most commonly employed internal standards in both GC/MS and LC/MS methods due to their similar properties and absence in real samples. As for the other detection techniques, a rapid Biacore biosensor immunoassay of NP was developed, and two types of antibodies were used (Samsonova et al. 2004): polyclonal antibodies with high cross-reactivity to technical NP and a monoclonal antibody very specific to 4-n-NP. 9-(p-Hydroxyphenyl) nonanoic acid were immobilized onto the surface of a sensor chip. The best assay sensitivity was achieved using a flow rate of 50 μl/min and an injection time of 2 min. For the assay incorporating monoclonal antibodies, a limit of detection of 2 ng/mL for 4-n-NP was achieved. With polyclonal antibodies,

one order of lower sensitivity was observed for the target compound. Applicability of the developed assays to ecological monitoring was tested in experiments using shellfish samples. In the assay using the monoclonal antibody specific to 4-n-NP, 31 shellfish samples were found to be negative.

References Abend, A.M., Chung, L., McCollum, D.G., Wuelfing, W.P. 2003. Development and validation of an automated extraction method (accelerated solvent extraction and a reverse-phase HPLC analysis method for assay of ivermectin in a meat-based chewable formulation. Journal of Pharmaceutical and Biomedical Analysis 31(6):1177–1183. Ademollo, N., Ferrara, F., Delise, M., Fabietti, F., Funari, E. 2008. Nonylphenol and octylphenol in human breast milk. Environment International 34(7):984– 987. Ahel, M., Giger, W. 1993a. Aqueous solubility of alkylphenols and alkylphenol polyethoxylates. Chemosphere 26(8):1461–1470. Ahel, M., Giger, W. 1993b. Partitioning of alkylphenols and alkylphenol polyethoxylates between water and organic-solvents. Chemosphere 26(8):1471–1478. Ahel, M., Giger, W., Koch, M. 1994a. Behavior of alkylphenol polyethoxylate surfactants in the aquatic environment. I. Occurrence and transformation in sewage-treatment. Water Research 28(5):1131–1142. Ahel, M., Giger, W., Schaffner, C. 1994b. Behavior of alkylphenol polyethoxylate surfactants in the aquatic environment. II. Occurrence and transformation in rivers. Water Research 28(5):1143–1152. Arukwe, A., Forlin, L., Goksoyr, A. 1997. Xenobiotic and steroid biotransformation enzymes in Atlantic salmon (Salmo salar) liver treated with an estrogenic compound, 4-nonylphenol. Environmental Toxicology and Chemistry 16(12):2576–2583. Barker, S.A., Long, A.R., Short, C.R. 1989. Isolation of drug residues from tissues by solid phase dispersion. Journal of Chromatography 475:353–361. Barker, S.A., Long, A.R., Hines, M.E. 1993. Disruption and fractionation of biological materials by matrix solid-phase dispersion. Journal of Chromatography A 629(1):23–34. Barker, S.A. 2007. Matrix solid phase dispersion (MSPD). Journal of Biochemical and Biophysical Methods 70(2):151–162. Benijts, T., Dams, R., Lambert, W., De Leenheer, A. 2004. Countering matrix effects in environmental liquid chromatography-electrospray ionization tandem mass spectrometry water analysis for endocrine disrupting chemicals. Journal of Chromatography A 1029(1–2):153–159. Blackburn, M.A., Kirby, S.J., Waldock, M.J. 1999. Concentrations of alkyphenol polyethoxylates enter-

Surfactants

ing UK estuaries. Marine Pollution Bulletin 38(2):109–118. Burkhardt-Holm, P., Wahli, T., Meier, W. 2000. Nonylphenol affects the granulation pattern of epidermal mucous cells in rainbow trout, Oncorhynchus mykiss. Ecotoxicology and Environmental Safety 46(1):34–40. Cai, Y.Q., Jiang, G.B., Liu, J.F., Liang, X., Yao, Z.W., Liu, J.M., Liu, J.Y., Zhou, Q.X. 2004. Solid-phase microextraction coupled with high performance liquid chromatography fluorimetric detection for the determination of bisphenol A, 4-n-nonylphenol, and 4-tert-octylphenol in environmental water samples. Analytical Letters 37(4):739–753. Carabias-Martinez, R., Rodriguez-Gonzalo, E., RevillaRuiz, P. 2006. Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography-mass spectrometry: Study of background contamination. Journal of Chromatography A 1137(2):207–215. Casajuana, N., Lacorte, S. 2004. New methodology for the determination of phthalate esters, bisphenol A, bisphenol A diglycidyl ether, and nonylphenol in commercial whole milk samples. Journal of Agricultural and Food Chemistry 52(12):3702–3707. Chapin, R.E., Delaney, J., Wang, Y.F., Lanning, L., Davis, B., Collins, B., Mintz, N., Wolfe, G. 1999. The effects of 4-nonylphenol in rats: A multigeneration reproduction study. Toxicological Sciences 52(1): 80–91. Chen, M.L., Lee, H.Y., Chuang, H.Y., Guo, B.R., Mao, I.F. 2009. Association between nonylphenol exposure and development of secondary sexual characteristics. Chemosphere 76(7):927–931. Crouch, M.D.A., Barker, S. 1997. Analysis of toxic wastes in tissues from aquatic species: Applications of matrix solid-phase dispersion. Journal of Chromatography A 774(1–2):287–309. Danzo, B.J., Shappell, H.W., Banerjee, A., Hachey, D.L. 2002. Effects of nonylphenol, 1,1-dichloro-2,2-bis(pchlorophenyl)ethylene (p, p′-DDE), and pentachlorophenol on the adult female guinea pig reproductive tract. Reproductive Toxicology 16(1):29–43. Datta, S., Loyo-Rosales, J.E., Rice, C.P. 2002. A simple method for the determination of trace levels of alkylphenolic compounds in fish tissue using pressurized fluid extraction, solid phase cleanup, and highperformance liquid chromatography fluorescence detection. Journal of Agricultural and Food Chemistry 50(6):1350–1354. De Voogt, P., De Beer, K., Van der Wielen, F. 1997. Determination of alkylphenol ethoxylates in industrial and environmental samples. Trends in Analytical Chemistry 16(10):584–595. Dodds, E., Lawson, W. 1938. Molecular structure in relation to oestrogenic activity. Compounds without a phenanthrene nucleus. Proceedings of the Royal Society of London B 125:222–232. Drew, M. 2006. Surfactant Science and Technology, 3d ed. John Wiley/VCH: New York. Erickson, B.E. 2002. Endocrine-disrupting nonylphenols detected in wide range of foods. Environmental

321

Science & Technology 36(9):178A-179A. Ferrara, F., Fabietti, F., Delise, M., Bocca, A.P., Funari, E. 2001. Alkylphenolic compounds in edible molluscs of the Adriatic Sea (Italy). Environmental Science & Technology 35(15):3109–3112. Ferrara, F., Fabietti, F., Delise, M., Funari, E. 2005. Alkylphenols and alkylphenol ethoxylates contamination of crustaceans and fishes from the Adriatic Sea (Italy). Chemosphere 59(8):1145–1150. Ferrara, F., Ademollo, N., Delise, M., Fabietti, F., Funari, E. 2008. Alkylphenols and their ethoxylates in seafood from the Tyrrhenian Sea. Chemosphere 72(9): 1279–1285. Gabriel, F.L.P., Giger, W., Guenther, K., Kohler, H.P.E. 2005a. Differential degradation of nonylphenol isomers by Sphingomonas xenophaga Bayram. Applied and Environmental Microbiology 71(3): 1123–1129. Gabriel, F.L.P., Heidlberger, A., Rentsch, D., Giger, W., Guenther, K., Kohler, H.P.E. 2005b. A novel metabolic pathway for degradation of 4-nonylphenol environmental contaminants by Sphingomonas xenophaga Bayram-ipso-hydroxylation and intramolecular rearrangement. Journal of Biological Chemistry 280(16): 15526–15533. Gadzala-Kopciuch, R., Filipiak, A., Buszewski, B. 2008. Isolation, purification, and determination of 4-n-nonylphenol and 4-tert-octylphenol in aqueous and biological samples. Talanta 74(4):655–660. Giger, W., Brunner, P.H., Schaffner, C. 1984. 4-Nonylphenol in sewage sludge: Accumulation of toxic metabolites from nonionic surfactants. Science 225:623–625. Güenther, K., Durbeck, H.W., Kleist, E., Thiele, B., Prast, H., Schwuger, M. 2001. Endocrinedisrupting nonylphenols-ultra-trace analysis and time-dependent trend in mussels from the German bight. Fresenius Journal of Analytical Chemistry 371:782–786. Güenther, K., Heinke, V., Thiele, B., Kleist, E., Prast, H., Raecker, T. 2002. Endocrine disrupting nonylphenols are ubiquitous in food. Environmental Science & Technology 36:1676–1680. Gundersen, J.L. 2001. Separation of isomers of nonylphenol and select nonylphenol polyethoxylates by high-performance liquid chromatography on a graphitic carbon column. Journal of Chromatography A 914(1–2):161–166. Hemmer, M.J., Hemmer, B.L., Bowman, C.J., Kroll, K.J., Folmar, L.C., Marcovich, D., Hoglund, M.D., Denslow, N.D. 2001. Effects of p-nonylphenol, methoxychlor, and endosulfan on vitellogenin induction and expression in sheepshead minnow (Cyprinodon variegatus). Environmental Toxicology and Chemistry 20(2):336–343. Hu, J., Wan, Y., Shao, B., Jin, X., An, W., Jin, F., Yang, M., Wang, X., Sugisaki, M. 2005a. Occurrence of trace organic contaminants in Bohai Bay and its adjacent Nanpaiwu River, North China. Marine Chemistry 95(1–2):1–13. Hu, J.Y., Jin, F., Wan, Y., Yang, M., An, L.H., An, W., Tao, S. 2005b. Trophodynamic behavior of

322

Analysis of Endocrine Disrupting Compounds in Food

4-nonylphenol and nonylphenol polyethoxylate in a marine aquatic food web from Bohai Bay, North China: Comparison to DDTs. Environmental Science & Technology 39(13):4801–4807. Ieda, T., Horii, Y., Petrick, G., Yamashita, N., Ochiai, N., Kannan, K. 2005. Analysis of nonylphenol isomers in a technical mixture and in water by comprehensive two-dimensional gas chromatography-mass spectrometry. Environmental Science & Technology 39(18): 7202–7207. Inoue, K., Kondo, S., Yoshie, Y., Kato, K., Yoshimura, Y., Horie, M., Nakazawa, H. 2001. Migration of 4-nonylphenol from polyvinyl chloride food packaging films into food simulants and foods. Food Additives and Contaminants 18(2):157–164. Jin, F., Hu, J.Y., Yang, M. 2007. Vertical distribution of nonylphenol and nonylphenol ethoxylates in sedimentary core from the Beipaiming Channel, North China. Journal of Environmental Sciences 19(3):353–357. Jobling, S., Sheahan, D., Osborne, J.A., Matthiessen, P., Sumpter, J.P. 1996. Inhibition of testicular growth in rainbow trout (oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals. Environment Toxicology & Chemistry 15(2):194–202. Jonkers, N., Govers, H., De Voogt, P. 2005. Adduct formation in LC-ESI-MS of nonylphenol ethoxylates: Mass spectrometrical, theoretical, and quantitative analytical aspects. Analytica Chimica Acta 531(2): 217–228. Jonkers, N., Knepper, T.P., De Voogt, P. 2001. Aerobic biodegradation studies of nonylphenol ethoxylates in river water using liquid chromatography-electrospray tandem mass spectrometry. Environmental Science & Technology 35(2):335–340. Kannan, K., Keith, T.L., Naylor, C.G., Staples, C.A., Snyder, S.A., Giesy, J.P. 2003. Nonylphenol and nonylphenol ethoxylates in fish, sediment, and water from the Kalamazoo River, Michigan. Archives of Environmental Contamination and Toxicology 44(1): 77–82. Kawaguchi, M., Inoue, K., Sakui, N., Ito, R., Izumi, S., Makino, T., Okanouchi, N., Nakazawa, H. 2004. Stir bar sorptive extraction and thermal desorption-gas chromatography-mass spectrometry for the measurement of 4-nonylphenol and 4-tert-octylphenol in human biological samples. Journal of Chromatography B 799(1):119–125. Keith, T.L., Snyder, S.A., Naylor, C.G., Staples, C.A., Summer, C., Kannan, K., Giesy, J.P. 2001. Identification and quantitation of nonylphenol ethoxylates and nonylphenol in fish tissues from Michigan. Environmental Science & Technology 35(1):10–13. Kim, H.S., Shin, J.H., Moon, H.J., Kang, I.H., Kim, T.S., Kim, I.Y., Seok, J.H., Pyo, M.Y., Han, S.Y. 2002. Comparative estrogenic effects of p-nonylphenol by 3-day uterotrophic assay and female pubertal onset assay. Reproductive Toxicology 16(3):259–268. Kim, Y.S., Katase, T., Sekine, S., Inoue, T., Makino, M., Uchiyama, T., Fujimoto, Y., Yamashita, N. 2004. Variation in estrogenic activity among fractions of a commercial nonylphenol by high performance liquid chromatography. Chemosphere 54(8):1127–1134.

Klejdus, B., Mikelov, R., Adam, V., Zehnáek, J., Vacek, J., Kizek, R., Kuán, V. 2004. Liquid chromatographicmass spectrometric determination of genistin and daidzin in soybean food samples after accelerated solvent extraction with modified content of extraction cell. Analytica Chimica Acta 517(1–2):1–11. Kloepfer, A., Quintana, J.B., Reemtsma, T. 2005. Operational options to reduce matrix effects in liquid chromatography-electrospray ionisation-mass spectrometry analysis of aqueous environmental samples. Journal of Chromatography A 1067(1–2):153–160. Knepper T.P., Barcelo D., De Voogt P. 2003. Analysis and Fate of Surfactants in the Aquatic Environment, vol XL. Elsevier: Amsterdam. Kuch, H.M., Ballschmiter, K. 2001. Determination of endocrine-disrupting phenolic compounds and estrogens in surface and drinking water by HRGC-(NCI)MS in the picogram per liter range. Environmental Science Technology 35(15):3201–3206. Laws, S.C., Carey, S.A., Ferrell, J.M., Bodman, G.J., Cooper, R.L. 2000. Estrogenic activity of octylphenol, nonylphenol, bisphenol A, and methoxychlor in rats. Toxicological Sciences 54(1):154–167. Li, C.T., Cheng, C.Y., Ding, W.H. 2008. Determination of alkylphenol residues in baby-food purees by steam distillation extraction and gas chromatography-mass spectrometry. Food Chemistry and Toxicology 46(2): 803–807. Lietti, E., Marin, M.G., Matozzo, V., Polesello, S., Valsecchi, S. 2007. Uptake and elimination of 4-nonylphenol by the clam Tapes philippinarum. Archives Environmental Contamination and Toxicology 53(4):571–578. Lin, W.C., Wang, S.L., Cheng, C.Y., Ding, W.H. 2009. Determination of alkylphenol residues in breast and commercial milk by solid-phase extraction and gas chromatography-mass spectrometry. Food Chemistry 114(2):753–757. Liu, X., Zhang, X., Zhang, H., Liu, M. 2008. A chemometric strategy for optimization of solidphase microextraction: Determination of bisphenol A and 4-nonylphenol with HPLC. Journal of Chromatographic Science 46(7):596–600. Long, A.R., Hsieh, L.C., Malbrough, M.S., Short, C.R., Barker, S.A. 1990. Matrix solid phase dispersion (MSPD) extraction and liquid chromatographic determination of five benzimidazole anthelmintics in pork muscle tissue. Journal of Food Composition and Analysis 3(1):20–26. Loyo-Rosales, J.E., Rosales-Rivera, G.C., Lynch, A.M., Rice, C.P., Torrents, A. 2004. Migration of nonylphenol from plastic containers to water and a milk surrogate. Journal of Agricultural and Food Chemistry 52(7):2016–2020. Lu, Y.Y., Chen, M.L., Sung, F.C., Paulus Shyi-Gang, W., Mao, I.F. 2007. Daily intake of 4-nonylphenol in Taiwanese. Environment International 33(7):903– 910. Mao, I.F., Lu, Y.Y., Chen, M.L. 2006. A simplified method for simultaneous quantitation of alkylphenols and alkylphenol ethoxylates in meat and fish using high-performance liquid chromatography with

Surfactants

fluorescence detection. International Journal of Environmental Analytical Chemistry 86(10):713– 722. Matozzo, V., Gagé, F., Marin, M.G., Ricciardi, F., Blaise, C. 2008. Vitellogenin as a biomarker of exposure to estrogenic compounds in aquatic invertebrates: A review. Environment International 34(4):531–545. Matozzo, V., Marin, M.G. 2005. Can 4-nonylphenol induce vitellogenin-like proteins in the clam Tapes philippinarum? Environmental Research 97(1): 43–49. McLeese, D.W., Zitko, V., Sergeant, D.B., Burridge, L., Metcalfe, C.D. 1981. Lethality and accumulation of alkylphenols in aquatic fauna. Chemosphere 10(7): 723–730. Meier, S., Klungsoyr, J., Boitsov, S., Eide, T., Svardal, A. 2005. Gas chromatography-mass spectrometry analysis of alkylphenols in cod (Gadus morhua) tissues as pentafluorobenzoate derivatives. Journal of Chromatography A 1062(2):255–268. Moeder, M., Martin, C., Harynuk, J., Gorecki, T., Vinken, R., Corvini, P.F.X. 2006. Identification of isomeric 4-nonylphenol structures by gas chromatographytandem mass spectrometry combined with cluster analysis. Journal of Chromatography A 1102(1–2): 245–255. Mueller, G.C., Kim, U.H. 1978. Displacement of estradiol from estrogen receptors by simple alkyl phenols. Endocrinology 102:1429–1435. Muncke, J. 2009. Exposure to endocrine disrupting compounds via the food chain: Is packaging a relevant source? Science of the Total Environment 407(16): 4549–4559. Nielsen, T.K., Kryger, S. 1969. Determination of volatile oil in drugs by distillation in steam. Dansk Tidsskrift for Farmaci 43(2):39–44. Okayasu, Y., Komori, K., Suzuki, Y., Tanaka, H., Yasojima, M. 2005. Nonylphenol formation from nonylphenol ethoxylates in activated sludge process. Journal of Japan Society on Water Environment 28:671–676. Otaka, H., Yasuhara, A., Morita, M. 2003. Determination of bisphenol A and 4-nonylphenol in human milk using alkaline digestion and cleanup by solidphase extraction. Analytical Sciences 19(12):1663– 1666. Pan, Y.P., Tsai, S.W. 2008. Solid phase microextraction procedure for the determination of alkylphenols in water by on-fiber derivatization with N-tert-butyldimethylsilyl-N- methyltrifluoroacetamide. Analytica Chimica Acta 624(2):247–252. Pojana, G., Bonfa, A., Busetti, F., Collarin, A., Marcomini, A. 2004. Determination of natural and synthetic estrogenic compounds in coastal lagoon waters by HPLCelectrospray-mass spectrometry. International Journal of Environmental Analytical Chemistry 84(10): 717–727. Pojana, G., Gomiero, A., Jonkers, N., Marcomini, A. 2007. Natural and synthetic endocrine disrupting compounds (EDCs) in water, sediment, and biota of a coastal lagoon. Environment International 33(7): 929–936.

323

Rhind, S.M., Kyle, C.E., Telfer, G., Duff, E.I., Smith, A. 2005. Alkyl phenols and diethylhexyl phthalate in tissues of sheep grazing pastures fertilized with sewage sludge or inorganic fertilizer. Environmental Health Perspectives 113(4):447–453. Rudel, R.A., Camann, D.E., Spengler, J.D., Korn, L.R., Brody, J.G. 2003. Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other endocrinedisrupting compounds in indoor air and dust. Environmental Science & Technology 37 4543– 4553. Samsonova, J.V., Uskova, N.A., Andresyuk, A.N., Franek, M., Elliott, C.T. 2004. Biacore biosensor immunoassay for 4-nonylphenols: assay optimization and applicability for shellfish analysis. Chemosphere 57(8):975–985. Schmitz-Afonso, I., Loyo-Rosales, J.E., de la Paz Aviles, M., Rattner, B.A., Rice, C.P. 2003. Determination of alkylphenol and alkylphenolethoxylates in biota by liquid chromatography with detection by tandem mass spectrometry and fluorescence spectroscopy. Journal of Chromatography A 1010(1):25–35. Shao, B., Hu, J.Y., Yang, M. 2002. Determination of nonylphenol ethoxylates in the aquatic environment by normal phase liquid chromatography-electrospray mass spectrometry. Journal of Chromatography A 950(1–2):167–174. Shao, B., Han, H., Hu, J., Zhao, J., Wu, G., Xue, Y., Ma, Y., Zhang, S. 2005a. Determination of alkylphenol and bisphenol A in beverages using liquid chromatography/ electrospray ionization tandem mass spectrometry. Analytica Chimica Acta 530(2):245–252. Shao, B., Hu, J.Y., Yang, M., An, W., Tao, S. 2005b. Nonylphenol and nonylphenol ethoxylates in river water, drinking water, and fish tissues in the area of Chongqing, China. Archives of Environmental Contamination and Toxicology 48(4):467–473. Shao, B., Han, H., Li, D., Ma, Y., Tu, X., Wu, Y. 2007a. Analysis of alkylphenol and bisphenol A in meat by accelerated solvent extraction and liquid chromatography with tandem mass spectrometry. Food Chemistry 105(3):1236–1241. Shao, B., Han, H., Tu, X., Huang, L. 2007b. Analysis of alkylphenol and bisphenol A in eggs and milk by matrix solid phase dispersion extraction and liquid chromatography with tandem mass spectrometry. Journal of Chromatography B 850(1–2):412–416. Snyder, S.A., Keith, T.L., Naylor, C.G., Staples, C.A., Giesy, J.P. 2001a. Identification and quantitation method for nonylphenol and lower oligomer nonylphenol ethoxylates in fish tissues. Environmental Toxicology and Chemistry 20(9):1870–1873. Snyder, S.A., Keith, T.L., Pierens, S.L., Snyder, E.M., Giesy, J.P. 2001b. Bioconcentration of nonylphenol in fathead minnows (Pimephales promelas). Chemosphere 44(8):1697–1702. Soto, A.M., Justicia, H., Wray, J.W., Sonnenschein, C. 1991. p-Nonylphenol: An estrogenic xenobiotic released from “modified” polystyrene. Environmental Health Perspectives 92:167–173. Staples, C.A., Weeks, J., Hall, J.F., Naylor, C.G. 1998. Evaluation of aquatic toxicity and bioaccumulation of

324

Analysis of Endocrine Disrupting Compounds in Food

C8- and C9-alkylphenol ethoxylates. Environmental Toxicology and Chemistry 17(12):2470–2480. Thiele, B., Gunther, K., Schwuger, M.J. 1997. Alkylphenol ethoxylates: Trace analysis and environmental behavior. Chemical Reviews 97(8):3247– 3272. Tsuda, T., Takino, A., Kojima, M., Harada, H., Muraki, K. 1999. Gas chromatographic-mass spectrometric determination of 4-nonylphenols and 4-tert-octylphenol in biological samples. Journal of Chromatography B 723(1–2):273–279. Tsuda, T., Suga, K., Kaneda, E., Ohsuga, M. 2000a. Determination of 4-nonylphenol, nonylphenol monoethoxylate, nonylphenol diethoxylate, and other alkylphenols in fish and shellfish by high-performance liquid chromatography with fluorescence detection. Journal of Chromatography B 746(2):305–309. Tsuda, T., Takino, A., Kojima, M., Harada, H., Muraki, K., Tsuji, M. 2000b. 4-Nonylphenols and 4-tert-octylphenol in water and fish from rivers flowing into Lake Biwa. Chemosphere 41(5):757–762. Tsuda, T., Takino, A., Muraki, K., Harada, H., Kojima, M. 2001. Evaluation of 4-nonylphenols and 4-tertoctylphenol contamination of fish in rivers by laboratory accumulation and excretion experiments. Water Research 35(7):1786–1792. Tsuda, T., Suga, K., Kaneda, E., Ohsuga, M. 2002. 4-Nonylphenol, 4-nonylphenol mono- and diethoxylates, and other 4-alkylphenols in water and shellfish from rivers flowing into Lake Biwa. Bulletin of Environmental Contamination and Toxicology 68(1): 126–131. Uguz, C., Dagcan, A.T., Iscan, M., Togan, I. 2004. Accumulation of nonylphenol in gold fish and suckermouth catfish in the semi static aquarium system. Indian Veterinary Journal 81(5):581–583. Urraca, J.L., Marazuela, M.D., Moreno-Bondi, M.C. 2004. Analysis for zearalenone and α-zearalenol in cereals and swine feed using accelerated solvent extraction and liquid chromatography with fluorescence detection. Analytica Chimica Acta 524(1–2): 175–183. Veith, G.D., Kiwus, L.M. 1977. An exhaustive steamdistillation and solvent-extraction unit for pesticides and industrial chemicals. Bulletin of Environmental Contamination and Toxicology 17(6):631–636. Villagrasa, M., Guillamon, M., Eljarrat, E., Barcel, D. 2007. Matrix effect in liquid chromatographyelectrospray ionization mass spectrometry analysis of

benzoxazinoid derivatives in plant material. Journal of Chromatography A 1157(1–2):108–114. Vinatoru, M., Toma, M., Radu, O., Filip, P.I., Lazurca, D., Mason, T.J. 1997. The use of ultrasound for the extraction of bioactive principles from plant materials. Ultrasonics Sonochemistry 4(2):135–139. Vincent, M.D., Sneddon, J. 2009. Nonylphenol: An overview and its determination in oysters and wastewaters and preliminary degradation results from laboratory experiments. Microchemical Journal 92(1):112– 118. Wang, J., Dong, M., Shim, W.J., Kannan, N., Li, D. 2007. Improved cleanup technique for gas chromatographicmass spectrometric determination of alkylphenols from biota extract. Journal of Chromatography A 1171(1–2):15–21. Wenzel, A., Bohmer, W., Muller, J., Rudel, H., SchroterKermani, C. 2004. Retrospective monitoring of alkylphenols and alkylphenol monoethoxylates in aquatic biota from 1985 to 2001: Results from the German Environmental Specimen Bank. Environmental Science & Technology 38(6):1654–1661. White, R., Jobling, S., Hoare, S.A., Sumpter, J.P., Parker, M.G. 1994. Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135:175– 182. Yang, D.K., Ding, W.H. 2005. Determination of alkylphenolic residues in fresh fruits and vegetables by extractive steam distillation and gas chromatographymass spectrometry. Journal of Chromatography A 1088(1–2):200–204. Yang, L., Lan, C., Liu, H., Dong, J., Luan, T. 2006. Full automation of solid-phase microextraction/on-fiber derivatization for simultaneous determination of endocrine disrupting chemicals and steroid hormones by gas chromatography-mass spectrometry. Analytical and Bioanalytical Chemistry 386(2):391–397. Ying, G.G. 2006. Fate, behavior, and effects of surfactants and their degradation products in the environment. Environment International 32(3):417–431. Zhang, H., Zuehlke, S., Guenther, K., Spiteller, M. 2007. Enantioselective separation and determination of single nonylphenol isomers. Chemosphere 66(4): 594–602. Zhao, M., Van der Wielen, F., De Voogt, P. 1999. Optimization of a matrix solid-phase dispersion method with sequential clean-up for the determination of alkylphenol ethoxylates in biological tissues. Journal of Chromatography A 837(1–2):129–138.

Chapter 14 Polybrominated Biphenyls Antonia María Carro-Díaz and Rosa Antonia Lorenzo-Ferreira

Introduction

Historical perspective (1973–2009)

Brominated flame retardants (BFRs) are a group of chemical substances of anthropogenic origin that contain bromine. They are added to synthetic polymers (such as acrylonitrile-butadiene-styrene), treatments used in textiles, electronic equipment, and other materials to improve combustion resistance and to reduce toxic gas emissions (Georlette 2009). Additive type BFRs, such as the polybrominated biphenyls (PBBs), are added to polymers by means of physical mixtures, making it easy for them to be gradually released into the environment (Alaee et al. 2003). Owing to their lipophilic nature, they enter the trophic chain and are bioaccumulated in the blood, in breast milk, and in the fatty tissue of animals and humans. This is why the European Food Safety Authority (EFSA), in its recommendation of 2006 (pursuant to the follow-up of BFRs in food and animal feed), and the United Nations Environment Programme, through the Persistent Organic Pollutants Review Committee (POPRC 2007), highlight the importance of stepping up the control and study of these types of pollutants, including PBBs, in food and animal feed (EFSA 2006; UNEP 2006).

PBBs refer to a group of brominated hydrocarbons formed by substituting bromine atoms for hydrogen atoms in biphenyl (see Figure 14.1). PBBs were introduced as flame retardants in the early 1970s. They were added in the amount of 10% to synthetic fibers, molded thermoplastic parts, coverings, shellac varnishes, and electronic material (WHO 1994; Bailey et al. 2002). During the combustion process, PBBs act by eliminating free radicals, preventing the propagation of flames (Alaee et al. 2003). The estimated production of PBBs in the United States from 1970 to 1976 was 6000 tons (commercial quantities) (WHO 1994). In 1973 in the state of Michigan (USA), the accidental contamination of feed for dairy cattle, beef cattle, and poultry occurred, with a technical quality mixture of hexabromobiphenyl, THBB (FireMaster BP-6 or FF-1), which was mistakenly used instead of NutriMaster, a supplementary magnesium oxide cattle feed (de Wit 2002; Dunckel 1975). This incident served to shed light on the hazards posed by PBBs to the environment and human health. This led to the destruction of a large number of cattle, pigs, sheep, and chickens, as well as many different products made with derivatives of milk and eggs (Getty et al. 1977; Kay 1977; Di Carlo et al. 1978). The interest and general concern of the public were voiced in numerous publications, such as Environmental Health Perspectives. The journal’s April 1978 issue

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

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was devoted exclusively to PBBs. As a result of this accident, the production of THBB in the United States ceased in 1974, and it was finally prohibited in 1977 (Bailey et al. 2002). However, the U.S. production of technical quality mixtures of octabromobiphenyl, TOBB, and decabromobiphenyl, TDBB, continued until 1979, although the mixtures were primarily exported to Europe, where there are still several companies that produce these compounds (WHO 1994). The production of PBBs continued in Germany until 1985, and France manufactured TDBB until 2000 (WHO 1994; Hardy 2002). In 1979, Canada also prohibited all commercial uses of PBBs as well as their manufacture and processing (PNUMA 1992). EXIDIM, the European database on the importation and exportation of hazardous chemical products, established under the Rotterdam Agreement, received a total of six requests for the exportation of PBB (not included among these, however was hexabromobiphenyl) from 2003 to 2006 (one each in 2003 and 2004, and two in both 2005 and 2006). In this period, no PBB imports were recorded in the European Union (UNEP 2006). At the present time, the production of PBBs may still be underway in Asia (Lassen et al. 1999). The U.S. Environmental Protection Agency (EPA) requires notification by the international community of any manufacture or importation of PBBs so that there are no significant sources of these substances. Moreover, the use of hexabromobiphenyl is strictly regulated (EFSA 2006; UNEP 2006). Despite a reduction in the worldwide production, the presence of PBBs is still being detected in the environment and, therefore, may enter the trophic chain. The ubiquitous presence of PBBs has been documented in a broad range of samples (zooplankton, fish, birds, soils, sediments) (Luross et al. 2002; Herzke et al. 2005; de Wit et al. 2006; Hites 2006; Vetter et al. 2008a). The control measures adopted are based on the fact that the

long-distance transport mechanisms of PBBs in the atmosphere and their biotransport by marine birds are similar to those exhibited by polychlorinated biphenyls (PCBs) and DDT (Scheringer et al. 2006). PBBs accumulate in food chains, and there is evidence of their long-term toxicity in different species, appearing as endocrine disruptors, which are embryotoxic and teratogenic (NTP 1992; ATSDR 2004; Evenset et al. 2005; Darnerud 2008; Shen et al. 2008).

Scope of the chapter Human and environmental exposure to PBBs may occur through product use or the recycling of plastics containing PBBs and after processing them at disposal sites. There are increasing concerns over the export of electronic waste, or e-waste, to developing countries for fear of widespread releases of PBBs, especially hexabromobiphenyl, during recycling operations. Children are exposed to PBBs in the same way as the population in general, mainly through the consumption of contaminated food. Exposure may also take place by means of the transfer of PBBs that have accumulated during the gestational period to the fetus through the placenta. Given that PBBs are lipophilic substances, they can accumulate in human milk and be transferred to the breastfeeding infant (Joseph et al. 2009; Wang and Needham 2007). The appearance of decabromobiphenyl (deca-BB) and hexabromobiphenyl (hexaBB) in samples of the sediment from the large rivers of western Europe (OSPAR 2001), in birds (Herzke et al. 2005; Jaspers et al. 2006) and fish from northern Europe (von der Recke and Vetter 2008b), marine mammals (von der Recke and Vetter 2007a), and shellfish (Fernandes et al. 2008; van Leeuwen and de Boer 2008) has been well documented. An examination of Environmental Health Criteria 152 (WHO 1994) shows that hexaBBs are the most toxic PBBs. Their presence

Polybrominated Biphenyls

in the environment is a consequence of their direct use as flame retardants and their extreme persistence; they are highly resistant to degradation in water, soil, and sediments, both under lab conditions and in the field (UNEP 2006). The deca-BBs are very stable; however, there are studies that point to the existence of processes of metabolic debromination of PBBs with a high degree of bromination in living organisms (WHO 1994). Other degradation transformations occur through the action of ultraviolet light and through microbial debromination processes in anaerobic environments (OSPAR 2001). Therefore, these observations suggested that more attention should be given to the potential for dietary intake and occupational exposure to PBBs present in the environment. This chapter brings together all the information available on the properties and toxicological effects, transformations, state-of-the-art analytical methodology (methods of sample preparation and determination), incidence and

Structure and properties It is believed that many PBBs, PCBs, and other halogenated aromatic hydrocarbons share a common mechanism of action that is closely linked to similarities in their structural configuration, physicochemical properties, use, manufacture, methods of contamination, and toxicological impact. However, their differences may affect the relative bioavailability, absorption, accumulation of tissues, interaction with receptors, and the toxicity of the chemical substances (Hardy 2002). The structure and general molecular formula are given in Figure 14.1. As can be deduced from the structure, there are 209 congeners with molecular weights ranging from 233 to 943 uma. Ten homologous groups are classified according to the

Br

Br

Br

Br

Br

Br

Br

Br

2,2',3,3',4,4',5,5',6-Nonabromobiphenyl; PBB 206

Br

Br

2,2',3,3',4,4',5,5'-Octabromobiphenyl; PBB 194

Br

Br

Br

Br

2,2',4,4',5,5'-Hexabromobiphenyl; PBB 153

Br

Br

Br

Br

Br

Br

Br

Br

exposure, and legislation dealing with PBBs in foodstuffs or products related to the food chain.

Br

Br

327

Br

Br

Br

Br

Br

Br

Br

Br

Br

2,2',3,3',4,4',5,5',6,6’-Decabromobiphenyl; PBB 209

Figure 14.1. General chemical structure of PBBs and the most abundant congeners in the technical quality mixtures: THBB (PBB 153), TOBB (PBB 194 and PBB 206), and TDBB (PBB 209).

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Analysis of Endocrine Disrupting Compounds in Food

number of bromine atoms (monobrominated to decabrominated), and each homologous group contains one or more isomers. The position and degree of bromination give rise to structural differences that have a direct effect on the toxicity of the compounds (ATSDR 2004). Three technical quality mixtures containing a limited number of congeners were produced and distributed (WHO 1994; de Boer et al. 2000): technical hexabromobiphenyl (THBB No CAS 36355-01-8), technical octabromobiphenyl (TOBB No CAS 27858-07-7), and technical decabromobiphenyl (TDBB No CAS 13654-09-6) with bromine contents of 76%, 81%, and 85%, respectively (see Figure 14.1; Brinkman and de Kok 1980). THBB has been commercialized under the trade names of FireMaster BP-6 and FireMaster FF-1. They contain different proportions of homologous PBBs ranging from dibromobiphenyl to octabromobiphenyl. FireMaster FF-1 is made from FireMaster BP-6, with the addition of 2% calcium polysilicate as an antilumping agent. PBB 153 (2,2′,4,4′,5,5′-hexabromobiphenyl) is the most abundant congener in these mixtures (60%–80%), followed by PBB 180 (2,2′,3,4,4′,5,5′-heptabromobiphenyl) with an abundance of 12%–25% (Jansson et al. 1993; UNEP 2006). TOBB (e.g., Bromkal 80 and FR 250 14A) contains chiefly PBB 194 (2,2′,3,3′,4,4′,5,5′-octabromobiphenyl) and a high ratio (47%–60%) of PBB 206 (2,2 ′ ,3,3 ′ ,4,4 ′ ,5,5 ′ ,6 - nonabromobiphenyl) and PBB 208 (2,2′,3,3′,4,5,5′,6,6′nonabromobiphenyl), whereas octa-BBs, hepta-BBs, and PBB 209 (2,2′,3,3′,4,4′,5,5′, 6,6′-decabromobiphenyl) are present in lesser proportions. TDBB (e.g., Flammex B-10, HFO 101, and Adine 0102) contains mainly the congener PBB 209 (97%) (von der Recke and Vetter 2007b; Vetter et al. 2008a). PBBs are solids with boiling points ranging from 300°C to 568°C and low volatility (with vapor pressures between 0.27 and 3.2 × 10−10 Pa at 25°C). The solubility of these compounds in water decreases as the number of

bromine molecules increases, with values of n-octanol/water partition coefficient (log Kow) between 4.6 and 9.4. (SciFinder 2009; Kristoffersen et al. 2002; Darnerud 2008). They are liposoluble, with varying levels of solubility in different organic solvents. They are thermally labile and may photodegrade in the environment (Hill et al. 1982; ATSDR 2004).

Mechanisms and transformations The technique used to produce PBBs is based on a Friedel-Crafts reaction in which the biphenyl reacts with bromide in the presence of chloride in an organic solvent, using aluminum chloride, aluminum, or iron bromide as a catalyzer (Stepniczka 1976; Brinkman and de Kok 1980). Unlike what occurs with PCBs, technical mixtures of PBBs contain limited quantities of congeners (von der Recke and Vetter 2007b). Synthesized deca-BB, PBB 209 (degree of bromination/number of orthosubstituents, 10/4) contains <4% PBB 206 (9/3) and no other PBB. TOBB is composed of PBB 194 (8/2) and PBB 206 (9/3), as well as eight lower congeners from hepta-BB to deca-BB. As many as 44 congeners, from tri-BBs to octa-BBs, have been detected in THBB (von der Recke and Vetter 2007b). As compared with PCBs, BFRs may be more susceptible to degradation in the environment due to the fact that the carbonbromine link is weaker than the carbonchloride link. In the atmosphere, the two processes able to produce any noticeable degradation or transformation in PBBs are photooxidation by hydroxyl radicals (OH) and direct photolysis (UNEP 2006). The importance of the photochemical reaction in the presence of sunlight for the degradation or transformation of PBBs in the atmosphere cannot be assessed owing to the lack of available information (ATSDR 2004). PBBs are persistent in natural conditions on the ground, considering that the mean degradation period in the soil and sediments

Polybrominated Biphenyls

329

206 (9/3)

182 (7/3)

196 (8/3) 203 (8/4)

199 (8/3) 198 (8/3) 209 (10/4)

175 (7/3) 208 (9/4)

207 (9/4)

178 (7/3) 179 (7/4)

183 (7/3)

202 (8/4)

197 (8/4) 200 (8/4) 201 (8/4)

184 (7/4)

176 (7/4)

187 (7/3) 188 (7/4)

Figure 14.2. Scheme of nona-BBs and octa-BBs formed from the photolytic transformation of PBB 209 (10-/4). The bold and dotted lines indicate different ways of transforming nona-BBs and octa-BBs (in gray and bold, respectively) in hepta-BBs.

is >6 months (WHO 1994). Degradation may be attributed to processes of photodegradation, even though this process may only take place at or near the soil surface (ATSDR 2004). The biodegradation of PBBs in water under aerobic conditions is low, and although PBBs having a lower degree of substitution could be biodegraded in water and aerobic sediments, those with a higher degree of substitution are resistant to aerobic biodegradation (ATSDR 2004; UNEP 2006). Photolytic transformation Experiments have been conducted using different irradiation techniques that have produced the photolytic transformation of PBB 209 (10/4) in the same PBBs following similar debromination models (although after different periods of time) (von der Recke 2007b). In Figure 14.2, it is possible to see that the nona-BBs PBB 207 (9/4) and PBB 208 (9/4) have been obtained and identified, whereas PBB 206 (9/3)—the only nona-BB present in the technical mixtures—was not

observed in any of the experiments. Eight out of the possible 12 octa-BBs that exist in theory were formed during the photolytic transformation of PBB 209 (10/4). A longer irradiation time led to the appearance of BBs (brominated biphenyls) from penta-BB to nona-BB, with the identification of the heptaBBs PBB 176 (7/4), PBB 178 (7/3), and PBB 179 (7/4). Hexa-BBs were found after 30 minutes of irradiation, and penta-BBs after 60 minutes, whereas the nona-BBs were practically nonexistent. After a period of 180 minutes, only the presence of penta-BBs was detected and their concentrations had increased. The perbromination of a ring in some of the compounds may be the reason why no signs of the expected photodebromination are observed. The presence of di-ortho congeners and the debromination in two ortho- positions do not give rise to any of the expected photoproducts either (von der Recke and Vetter 2007b). During the photodebromination of PBB 209 (10/4), 13 out of the theoretically possible 42 hexa-BBs were detected. Only 4 of them

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Analysis of Endocrine Disrupting Compounds in Food

are found in the THBB mixture: PBB 149 (6/3), PBB 153 (6/2), PBB 154 (6/3), and PBB 135 (6/3). PBB 194 (8/2) produces three hexaBBs: PBB 153 (6/2), PBB 133 (6/2)—also formed from PBB 178 (8/3) and PBB 202 (8/4)—and PBB 146 (6/2), the only hexa-BB that can be formed from PBB 201 (8/4). In keeping with the results illustrated above, the elimination of Br from the ortho positions was not observed (von der Recke and Vetter 2007b). Anaerobic transformation of PBBs Although the degradation of PBBs by purely abiotic chemical reactions is considered to be highly unlikely (UNEP 2006), it has been demonstrated that the microorganisms found in fluvial sediments extracted from populated areas may biodegrade PBBs with a high degree of substitution, including mixtures of FireMaster, to form products with a lower bromine rate (Morris et al. 1992; ATSDR 2004). Experiments on the anaerobic transformation of TOBB with super-reduced cyanocobalamin (CCAs) at varying incubation times in fish tissue have been carried out (von der Recke and Vetter 2008a). After 6 h, the presence of hexa-BBs and an increase in hepta-BBs were detected. After 24 h, the principal components of the TOBB mixture were not detected. Tetra-BBs and tri-BBs were found after 3 and 7 days, respectively had elapsed. The simultaneous formation of penta-BBs and hepta-BBs proved that debromination was the main transformation process. The reductive debromination of PBBs does not occur mainly in the ortho positions. PBB 155 (6/4), PBB 136 (6/4), and PBB 148 (6/3) were able to be identified through the abiotic transformation of the fraction of TOBB containing PBB 209 (10/4) or from TDBB. These results confirm that the anaerobic transformation process of TOBB gives rise to important PBB congeners in the environ-

ment, but said PBBs are not found in the technical mixtures.

Toxicological effects The mechanism of action underlying several of the toxicological effects of PBBs, among which are the induction of metabolizing enzymes, immunotoxicity, hepatotoxicity, and reproductive toxicity, is thought to be due to an interaction with the Ah cell receptor (which is also the object of polychlorinated dioxins, furans, and dioxin type PCBs), which causes changes in gene expression (Capen 1992; Lambert et al. 1992; Chhabra et al. 1993; Henck et al. 1994; Hoque et al. 1998; Blanck et al. 2000a, b; Kristoffersen et al. 2002; ATSDR 2004; UNEP 2006). PBBs have been evaluated pursuant to the EU-Strategy for Endocrine Disrupters and have been classified as substances showing evidence of or a potential for being endocrine disruptors on the priority list of chemicals developed in accordance with the EU Strategy. Moreover, PBBs may promote genotoxic activity in other compounds by means of the activation of reactive intermediaries, owing to their enzymatic but not mutagenic induction properties (RanggaTabbu and Sleight 1992). In most of the studies on the toxicity of PBBs in animals, it was found to be produced by oral exposure. The semilethal dose (LD50) of FireMaster BP-6 administered orally in rats is 21.5 g/kg of body weight; the LD50 of TOBB administered orally in rats is >17 g/kg of body weight, and the DL50 of hexa-BB in rabbits by skin penetration is 5 g/kg of body weight (ATSDR 2004). Many toxicological studies have focused especially on PBB 153, the principal component of FireMaster. Moreover, the toxicity of FireMaster may depend on other components in the technical mixture: PBB 156, PBB 167, PBB 169, and PBB 118 (WHO 1994; Brown et al. 2004). Toxicological studies of low exposure levels of hexa-BBs in animals have been con-

Polybrominated Biphenyls

ducted for the characterization of the lowestobserved-adverse-effect-level (LOAEL) and non-observed-adverse-effects-level (NOAEL) of hexa-BBs (ATSDR 2004). Several chronic toxic effects produced by hexa-BBs have been observed in the liver and thyroid glands of the animals used for experimentation. Cancer-causing effects were seen with doses of 0.5 mg/kg of body weight per day, whereas no effects were observed after administering 0.15 mg/kg of body weight per day. Information on the effects of PBBs, including hexa-BBs, on human health comes from studies conducted on a human population in Michigan, where contaminated animal feed allowed these compounds to enter the human food chain. There is also information on the effects of PBBs on the health of workers exposed to the chemical product (Brown et al. 1981; Miceli et al. 1985), with a wide variety of symptoms being found (neurological and neuropsychiatric conditions, gastrointestinal, hepatic and skin problems, musculoskeletal disorders, effects on neurodevelopment, and immunological changes) (Di Carlo et al. 1978; Kimbrough 1987; ATSDR 2004). Exposure in utero takes place by the transfer of PBBs to the fetus through the placenta, and breastfeeding infants may also be exposed through the milk. The levels of PBB 153 found in human milk are 100 times higher than those encountered in the mother ’s blood (UNEP 2006). It has been suggested that there may be a relationship between the levels of PBBs found in serum and the risk of developing certain kinds of cancer, such as lymphoma and cancer of the digestive system (Hoque et al. 1998) or breast cancer (Henderson et al. 1995). In terms of toxicokinetics, and owing to the lipophilic nature of these compounds, the greatest relative quantity of PBBs, generally accumulates in the liver, fat, skin, and human breast milk. The PBB levels in both serum and adipose tissue are indicators of exposure to these compounds, and the simultaneous

331

control of PBBs in the two types of samples is more reliable (Anderson 1985). Hexa-BBs are bioaccumulative, with potentially harmful effects for human health, including carcinogenesis, reproductive toxicity, toxicity of the endocrine system and other hormones, even with very low levels of exposure. Data on their carcinogenicity are conclusive in animals, but not in humans. However, the International Agency for Research on Cancer (IARC) considered hexabromobiphenyl to be a potential carcinogen for human beings (IARC 1986).

Analytical methods Sample preparation techniques Over the last decade, the presence of brominated flame retardants in the environment has been recognized as a group of persistent pollutants. The growing concern over determining their presence in environmental and food samples is understandable given the wealth of articles that have been published, among which are a number of interesting reviews chiefly dedicated to the study of polybrominated diphenyl ethers (PBDEs) (Covaci et al. 2003; Stapleton 2006; D’Silva et al. 2004; de Boer et al. 2001). These reviews have recently been completed with a chapter on brominated flame retardants published in volume 51 of Wilson and Wilson’s Comprehensive Analytical Chemistry (Covaci et al. 2008a). This bibliography is the foundation of the description of the analytical methodology used in the determination of PBBs in foods. The similarity in the structure and properties of the different families of polyhalogenated biphenyls have led, in many cases, to the joint determination of PCBs, PBDEs, PBBs, and PBDD/Fs (polybrominated dioxins and furans) (Covaci et al. 2007). An overview of selected methods classified according to the sample treatment techniques used for the determination of PBBs in foods appears in Table 14.1.

332 Soxhlet, hexane/ acetone (1 : 1) 48 h Solid–liquid extraction. 300 mL DCM

17 PBBs (mono-, hexa-BBs) PBB 153 23 PBBs (1–209)

PBB 153

Breast milk

Fish, shellfish

Vegetables, pulses, rice, hen eggs, chicken, pork, fish Bird eggs

Shellfish

Trout

Mono-, di-, tri-, tetra-, penta- and hexa-BBs PBB 49, 52, 77

Solid–liquid extraction (Na2SO4 anh.) 250 mL acetone/ hexane (2 : 1) Soxhlet, hexane/ acetone (3 : 1) 6–12 h Soxhlet, 180 mL hexane/ acetone (3 : 1) 24 h

PBB 153

Breast milk

Water

Solid–liquid extraction. 200 mL DCM + silica gel acid

SPME (PDMS fiber) 30 min MSPD (DE) 12 mL DCM

SBSE 5 h

PBB 7, 31, 103, 153 PBB 15, 49

Water

Extraction

Analytes

Matrix

Multilayer column (Na2SO4 anh. + acidic silica + Basic silica + Na2SO4 anh.)/ Carbon column. Hexane + DCM/ hexane + toluene. Acidic silica + alumina columns

GPC. 300 mL DCM/ hexane (1 : 1) + silica gel/alumina column

GPC + silica column chromatography + acid sulphuric Multilayer silica gel column (Na2SO4 anh./ AgNO3/silica/acidic silica/silica/Na2SO4 anh.) 100 mL hexane + DCM: hexane GPC/SPE (Isolute)

SPE, two-layered (silica and silica/sulfuric acid). Hexane GPC + SPE (alumina/ Na2SO4 anh./Florisil/ silica gel/Na2SO4 anh.) toluene

—

—

Cleanup

Table 14.1. Selected methods to determine PBBs in food samples.

GC-EI-HRMS

GC-EI-HRMS

n.r.

60–120

n.r.

n.r.

GC-EI-MS

GC-EI-HRMS

n.r.

n.r.

GC-EI-HRMS

GC-ECNI-MS

74

90–97

82.9–100.1

Recoveries (%)

GC-EI-MS/ MS GC-EI-HRMS

GC-EI-MS

Determination

n.r.

0.01– 0.04 ng/g ww

n.r.

0.08– 0.24 ng/g dw

n.r.

n.r.

0.1 ng/g of lipid

0.4– 3.5 ng/L 9–7.5 pg/L

LODs

Fernandes et al. 2004, 2008, 2009

Luross et al. 2002

Gao et al. 2009

van Leeuwen and de Boer 2008 Zhao et al. 2009

Sjödin et al. 2004; Focant et al. 2004 Shen et al. 2008

Prieto et al. 2008 Polo et al. 2004

References

333

PBB 101, 118, 138, 149, 153, 156, 157, 167

Cheese, milk, fish

MSPD (20 g silica gel/ Na2SO4 anh. (1 : 1)) 400 mL acetone/ hexane (1 : 1)

MAES 15 mL hexane + 9 mL 1M KOH in MeOH 3 min MAE 15 mL hexane, 9 min MAE(14 mL hexane, 15 min)-SPME (PDMS fiber) 75°C, 60 min MSPD (1 g C18) 30 mL hexane

Supercritical fluid extraction (SFE) 27 min SFE-SPME (PDMS fibre) 75°C, 60 min ASE acetone/ cyclohexane (1 : 3) 15 min

Extraction

Two layered (acidic silica + alumina) integrated in MSPD Multilayer column (silica neutra/acidic silica/ basic silica) 125 mL hexane + SPE (Envicarb)

SPE (acidic silica column) 15 mL hexane SPE (acidic silica column) 15 mL hexane

SPE (acidic silica column) 15 mL hexane

Integrated in SFE chamber (acidic silica + basic alumina) Integrated in SFE chamber (acidic silica + basic alumina) GPC + Florisil column 35 mL hexane/toluene

Cleanup

72–116.6

GC-μECD

PTV-GC-MS/ MS

GC-EI-MS/ MS

n.r.

72.4–85

n.r.

78.3–82.9

GC-μECD

GC-EI-MS/ MS

n.r.

69–102

89–106

Recoveries (%)

GC-EI-MS GC-ECNIMS/MS

GC-EI-MS/ MS

GC-EI-MS/ MS

Determination

DE, diatomaceous earth; GPC, gel permeation chromatography; DCM, dichloromethane; n.r., not reported.

PBB 15, 49

PBB 15, 49

PBB 15, 49

Fish feed, shellfish

Fish feed, shellfish Fish feed, cockle

Fish feed, shellfish

PBB 15, 49

Fish feed, turbot, shellfish Bird eggs THBB, TOBB, Di-, tri-, tetra-, penta-, hexa-, hepta-BBs PBB 15, 49

PBB 15, 49

Analytes

Fish feed, shellfish

Matrix

n.r.

02–03 ng/g

0.04– 0.4 ng/g 50–80 pg/g

0.04– 0.4 ng/g

n.r.

1.2–2.1 pg/g

0.13– 0.25 ng/g

LODs

Gomara et al. 2006

Carro et al. 2005

Fajar et al. 2008 Carro et al. 2007

Fajar et al. 2008

Vetter et al. 2008a; Herzke et al. 2005; Götsch et al. 2005

Rodil 2005b

Rodil et al. 2007

References

334

Analysis of Endocrine Disrupting Compounds in Food

Liquid matrices The low concentrations of BFRs present in water make the development of sensitive trace analysis methods necessary. A method involving stir bar sorptive extraction (SBSE), followed by thermal desorption of PBBs (PBB 7, PBB 31, PBB 103, PBB 153) was optimized using the design of experiments in water samples. The factors affecting the efficiency of SBSE were, e.g., sample volume, 20 mL. The experimental parameters were fixed at 10 minutes and a desorption flow of 50 mL/min with a vent pressure of 7 psi. When the method was applied to six water samples, concentrations in the 9–13 ng/L range were found (Prieto et al. 2008). Solid-phase microextraction (SPME) is a rapid and simple analytical technique that uses coated fused-silica fibers to extract trace concentrations of analytes (PBBs and PBDEs) from aqueous samples (Polo et al. 2004). The factors affecting the extraction process have been studied in detail with the experimental design. The extraction conditions finally selected for all of the compounds require the use of PDMS (polydimethylsiloxane) fibers, 10 mL sample volume, sampling headspace, 100°C extraction temperature, stirring, and 30 min extraction time. The salt addition is not necessary. The microextraction method was validated by evaluating the linear range, repeatability, accuracy, and limits of detection. In the determination of PBB 15 and PBB 49, the linear range stays between 1 and 500 pg/mL, with limits of detection of 9 and 7.5 pg/L, respectively, and recoveries of over 90%. The PBB residues in drinking water were extracted by SPE cartridges packed with C18 sorbent, following the modified method 3535a (EPA 2007). The SPE cartridges were pre-eluted with methylene chloride and activated with methanol and water. After water extraction, the analytes were eluted with three portions of 5 mL each, hexane, hexane/ methylene chloride (9 : 1 v/v), and hexane/

methylene chloride (1 : 1 v/v). The eluates were dried, concentrated and redissolved in 200 μL hexane (Zhao et al. 2009). Human milk is used in biomonitoring studies to determine the exposure of the nursing child to BFRs. A semiautomatic method, based on the dispersion of the milk sample in a solid phase of diatomaceous earth, was proposed by Sjödin et al. (Sjödin et al. 2004; Focant et al. 2004). The extraction procedure includes drying the sample and eluting target analytes and lipids with dichloromethane. To remove the coextracted lipids, the extracts underwent a cleanup procedure by SPE cartridge packed with silica and silica/sulfuric acid. A solid–liquid method based on homogenization of human milk with Na2SO4 and sea sand, and extraction with 250 mL acetone/hexane (2 : 1 v/v) has been applied by Shen et al. (2008). The cleanup was done in gel permeation chromatography and SPE. Solid matrices The traditional technique used in the preparation of solid samples is Soxhlet extraction with organic solvents, considered to be the method of reference. In order to determine BFR contamination (joint determination of PBB 153 and PBDE 154) in freshwater fish, marine fish and shellfish, and their exposure in the diet, van Leeuwen and de Boer (2008) analyzed 44 samples of different origin. The samples were extracted by means of Soxhlet using hexane/acetone (3 : 1) (de Boer et al. 2001). A study was conducted recently to estimate the total daily dietary intakes (TDIs) of PBBs in residents living close to e-waste disassembly sites (Zhao et al. 2009). Solid samples of vegetables, rice, pulse, chicken, pork, and fish were extracted in Soxhlet with the same solvent mixture. The concentrated extracts were purified in a multilayer silica gel column on which the different fractions with hexane : methylene chloride (90 : 10) were eluted. The low-brominated PBBs were

Polybrominated Biphenyls

those that contributed to the diet, with an estimated ingestion of 385.5 ng/day. However, PBB 209 was not detected in the food analyzed. A significant number of research works have confirmed that bird eggs can be used as effective indicators of anthropogenic pollution. They have been used to monitor and measure the levels and effects of different kinds of persistent organic pollutants (POPs). Gao et al. (2009) used Soxhlet extraction to evaluate the presence of PBB 153 and other organohalogen compounds in bird egg samples in North China. The procedure requires large amounts of organic solvents (n-hexane/acetone 3 : 1), an extraction time of 48 hours, and gel permeation chromatography for lipid removal and fractionation/ cleanup by SPE. As an alternative to classic Soxhlet extraction, other authors have proposed solid– liquid extraction using open column packing. The sample is mixed in a mortar with anhydrous sodium sulfate, transferred to an open column, and eluted with large volumes of solvents. The PBBs were separated and determined in lake trout after extraction and purification by gel permeation chromatography for bulk lipid removal (Luross et al. 2002). Recently, several methods of extraction and cleanup, based on solid–liquid extraction have been developed to assess the presence of PBBs and other organohalogenated pollutants in shellfish and to estimate exposure in the human diet (Fernandes et al. 2004, 2008, 2009). In these investigations, the sample was mixed with the solvent (200 mL hexane) and acidic silica gel (75 g) and transferred to a multilayer column and to another activated carbon column connected in series. The analytes were extracted and fractionated using organic solvent mixtures (hexane 100 mL and dichloromethane/ hexane 400 mL). The fraction containing PBBs was later purified using an acid treatment and adsorption chromatography to

335

obtain suitable extracts to be analyzed by high-resolution GC-MS. As mentioned above, classic extractions are time consuming, use large quantities of solvents, and require successive cleanup stages. Therefore, other alternative techniques have been developed to prepare food samples based on the use of compressed fluids as extracting agents (Mendiola et al. 2007). In supercritical fluid extraction (SFE), owing to its low polarity, supercritical carbon dioxide is suitable for the extraction of nonpolar compounds such as PBBs in food samples with considerable fat content. Rodil et al. (2007) developed a SFE-based multiresidue analytical methodology for the determination of OCPs, PCBs, PBBs (PBB 15 and PBB 49), and PBDEs from marine biological samples. In this work, various fat retainers were tested, and SFE parameters were optimized by means of the Doehlert design after the significant factors were identified by a screening study. The final experimental conditions were found by using desirability function optimization (60°C, 165 bar with a flow of 2 mL/min of CO2 for 5 min static extraction and 27 min dynamic extraction). Fat interferences were eliminated by means of a combination of 1.5 g of acidic silica and the same amount of aluminum oxide. Under these conditions, the fat remaining in the extracts was below 0.1%. Rodil (2005b) improved the analytical procedure described above by including a SPME step after the supercritical extraction to determine the same organohalogenated pollutants from fish feed and cultured marine species. This methodology offers the efficiency, rapidity, and selectivity of SFE combined with the increased sensitivity provided by the SPME. The optimization of the SFE/ SPME sample treatment was undertaken by an experimental design approach. Both methods were then applied to real aquaculture samples, including trout and turbot feed, turbot, cockles, clams, and mussels. PBB 15 was detected in the turbot and cockle (0.04

336

Analysis of Endocrine Disrupting Compounds in Food

and 0.2 ng/g, respectively). Detection limits were improved when SPME was used, reaching the picogram per gram level for all studied compounds. Pressurized liquid extraction (PLE) or accelerated solvent extraction (ASE) is based on the use of solvents at high temperatures (50–200°C) and pressures (500–3000 psi), significantly reducing extraction time and solvent volume. This technique has been used in recent years in PBB analysis (Vetter et al. 2008a; Herzke et al. 2005; Götsch et al. 2005). The extraction conditions were adapted from an early work by Herzke and colleagues (2002) designed to determine organochlorinated compounds. The homogenized samples were mixed with a 10-fold amount of dry Na2SO4 and extracted with a cyclohexane/ acetone mixture (v/v 3 : 1) at 100°C within 15 min (10 min static and 5 min heating time under a pressure of 10 Mpa). The separation of the lipids from the extracts obtained with ASE was carried out using gel-permeation chromatography and additional fractionation on a Florisil column. The evaluation of the PBBs found in the egg samples indicates that the dominant congeners were PBB 153 (present in the technical hexa-BB mixture), PBB 154 and PBB 155 (formed by the reductive debromination of deca-BB) (Vetter et al. 2008b). In their study, Götsch et al. (2005) demonstrated the possibility of the enantioselective separation of axially chiral PBBs 132 and 149 by a combination of HPLC fractionation of the atropisomers followed by GC-MS quantification of the volume-adjusted fractions. Microwave-assisted extraction (MAE) is a technique that has proven to be ideal in the extraction of different residues in food by means of microwave energy as a heat source. The extraction efficiency of the analyte of the food matrix depends on the following parameters that must be optimized: extraction temperature and time in addition to the nature and volume of the extraction solvent. Moreover, for lipid separation, the analysis

of brominated pollutants in food requires a purification stage in addition to the MAE, which may be performed by destructive or nondestructive methods (Covaci et al. 2008b). The efficiency of microwave-assisted extraction with saponification (MAES) for the determination of PBB 15 and PBB 49 and PBDEs in aquaculture samples is described and compared with microwave-assisted extraction (MAE) by Fajar et al. (2008). Chemometric techniques based on experimental designs and desirability functions were used for simultaneous optimization of the operational parameters used in the MAE and MAES processes. The optimal MAES conditions were 65°C and 3 min of extraction, and 15 mL hexane and 9 mL of 1 M KOH in methanol for saponification. With the optimized parameters, the lipid content of the extracts after SPE (with 3 g acidic silica gel) was less than 0.06% and 0.04% of the original lipid content as measured gravimetrically when MAE (18%) or MAES (0.7%) were applied, respectively. An effective and suitable combination of MAE and SPME was proposed to enhance sensitivity and selectivity for the quantitative GC analysis of a group of 15 pollutants, including PBBs in aquaculture solid samples (Carro et al. 2007). The initial MAE stage, carried out at 85°C, requires less than 1.5 g of raw sample and is both fast (15 min) and sparing in its use of solvent (14 mL of 1 : 1 hexane/dichloromethane). Subsequent SPE on acidic silica gel reduces lipid content to less than 0.05%, and further cleanup by HSSPME (headspace solid phase microextraction) (60 min at 75°C with a 100 μm PDMS-coated fused silica fiber) requires no additional solvent. The method has good precision, accuracy, and linearity with limits of quantification of 260 and 160 pg/g for PBB 15 and PBB 49, respectively. Matrix solid-phase dispersion (MSPD) is a sample-preparation technique with increasing acceptance in extraction and fractionation of a large number of organic compounds from

Polybrominated Biphenyls

solid, semisolid, and liquid matrices. Low sample and solvent consumption, straightforward application, and reduced cost, and the ability to simultaneously perform extraction and cleanup in a single step, are some of its major advantages (García-López 2008). A method based on MSPD-GC-MS/MS for the trace analysis of 15 polychlorinated and polybrominated compounds from aquaculture feed and cultured marine species has been developed by Carro et al. (2005). Spiked sample (1.5 g) and C18 (1 g) were placed in a glass mortar. A syringe barrel, containing a frit at the bottom, was filled (from bottom to top) with 2 g acidic silica, 1.5 g alumina (as cleanup adsorbents), and the homogenized matrix in sandwich mode, using another frit at the top of the column as a retainer. The column was then eluted with 30 mL hexane, which was concentrated to dryness by nitrogen blowdown concentrator, and the residue was finally redissolved in 200 μL hexane. Recoveries of PBB 15 and PBB 49 were 85% and 72.4%, respectively. Gomara et al. (2006) describe the fractionation of PCBs and BFRs, among which are included different PBBs (101, 118, 138, 149, 153, 156, 157, 167), in food samples (cheese, milk, horse mackerel) by MSPD. The lyophilized (freeze-dried) sample (5– 10 g) was homogenized with 20 g of silica gel mixture: anhydrous sodium sulfate 1 : 1 (w/w) transferred onto a column and extracted with 400 mL of 1 : 1 (v/v) acetone/hexane. The extract was purified for lipid removal by using acidic and basic modified silica gel multilayer columns, with hexane as the elution solvent. Final fractionation among the studied compounds was achieved by using SupelcleanT Supelco ENVI-Carb SPE cartridges (Bordajandi et al. 2003).

Determination techniques As mentioned above, the separation, identification, and quantification of PBBs, PCBs, and PBDEs are carried out jointly in a very

337

similar way by means of chromatographic techniques (Zhao et al. 2005). The 1970s witnessed the development of the first methods for the determination of PBBs based on gas chromatography/electron capture detection (GC-ECD) on packed columns. At the present time the GC-ECD system with capillary columns is still used to determine PBBs in human serum (Givens et al. 2007). GC-μECD has also produced satisfactory results in multiresidue separation, which includes PBBs, with detection limits ranging between 0.04 and 0.4 ng/g in aquaculture samples (Rodil et al. 2005a; Fajar et al. 2008). Appearing later were the first methods for the determination of PBDEs by means of gas chromatography–mass spectrometry detection (GC-MS) with capillary columns (ATSDR 2004; Covaci et al. 2008a,b; Zhao et al. 2008), which have also been adopted for the analysis of PBBs. Although structurally speaking there are 209 PBB congeners, only a small number are found in the technical mixtures. However, the profiles of the congeners found in food of animal origin coincide with those in the technical mixtures only to a limited extent, probably due to selective captation, metabolism, and degradation. PBB 153 is often detected along with PBDEs, coeluting with PBDE 154. The physicochemical properties of PBBs determine the way in which the sample will be introduced into the gas chromatography system. The most common injection systems are split/splitless, on-column, and injection with a programmed-temperature vaporizer (PTV). Each of the above injection methods has its advantages and its disadvantages, mainly related to availability, price, acceptable detection methods, and the discrimination of the congeners depending on molecular weight (Covaci et al. 2008a). A single capillary column of GC can provide sufficient resolution for the specific determination of congeners. Hence, this requires the use of relatively long columns (30–50 m) with small

338

Analysis of Endocrine Disrupting Compounds in Food

diameters (≤0.25 mm). By using columns with a narrow diameter (interior diameter = 0.1mm), it is possible to obtain a good resolution (Covaci and Dirtu 2008b). MS detectors are more selective than ECD for PBBs and PBDEs because they are able to identify and determine, on an individual basis, homologous compounds that can coeluate on high-resolution gas chromatography columns (HRGC). Electron impact ionization (EI) and electron capture negative ionization (ECNI) have been applied in MS to the simultaneous determination of PBBs and PBDEs in marine animal samples (de Boer et al. 1998). This method is advantageous in that it offers high sensitivity for compounds with more than four bromine atoms. The sensitivity is roughly 10 times greater than what is obtained with ECD. The low-resolution mass spectrometry (LRMS) techniques are most often based on ECNI, which offers higher sensitivity than EI. ECNI is a soft ionization technique that takes advantage of interactions between electrons of thermal energy and electrophilic molecules such as PBBs. In ECNI, low-energy electrons (thermal electrons) are generated by interactions between a high-energy electron beam and a gas, generally methane, and they react with analytes to form negative ions. The energy required to capture electrons depends on the molecular structure of the analytes. Some of the benefits of ECNI are efficient ionization, higher sensitivity, and less fragmentation than EI or positive chemical ionization (CI) (Covaci et al. 2003). However, the EI mode offers a wider range of possibilities in the selection of the ion as compared with the ECNI mode. The biomagnification of pollutants such as PBBs has been found all along the food chain in biota samples from Norwegian lakes by applying the GC-ECNI-LRMS technique (Evenset et al. 2005). HRGC combined with high-resolution mass spectrometry (HRMS) and the EI mode

has been used in the identification and quantification of PBBs in trout from the Great Lakes, using on-column injection (Luross et al. 2002), as well as in the determination of PBBs in human milk (Shen et al. 2008). Recently, GC-EI-HRMS with the selected reaction monitoring (SRM) mode has been the methodology applied in the study of brominated and chlorinated pollutants (PBBs among other BFRs) in mussels, oysters, and scallops in order to establish current levels and estimate human exposure through food (Fernandes et al. 2008). GC-EI-HRMS, with isotopic dilution has also been used in the analysis of samples of human milk in control studies to assess the exposure of breastfeeding infants to PBBs (Sjödin et al. 2004). In some cases, the complexity of real samples (food, biological, and environmental) requires a higher degree of selectivity than that which can be attained by means of EI-MS (Salgado-Petinal 2006a,b). Ion trapMS/MS has been used in the separation and selective determination of different pollutants, including PBBs, in samples of drinking water and wastewater at trace levels of picogram per liter (Polo et al. 2004). Several studies have made use of tandem MS coupled to GC to analyze PBBs, PBDEs, and PCBs in feeds used in aquaculture and in marine species (fish and shellfish) by means of different sample preparation techniques described in detail in the previous section. All of these studies reported low limits of detection (LODs) of around 0.1–0.3 ng/g (Carro et al. 2005), 1.2 and 2.1 pg/g (Rodil 2005b), 0.13–0.25 ng/g (Rodil et al. 2007), and 50– 80 pg/g (Carro et al. 2007). GC-ECNI-MS is considered to be a more sensitive method for the detection of PBBs. It has been used in the simultaneous analysis of several BFRs and PCBs in seagull eggs to find out whether these pollutants may play a role in the decline of the population of this species (Braune et al. 2007). This system was also used to determine the level of contamination with BFRs (including PBB 153) in fish

Polybrominated Biphenyls

and shellfish regularly consumed by the Dutch population (van Leeuwen and de Boer 2008). Both studies reported the coelution of PBB 153 with PBDE 154, using different GC-columns (HP-5MS and CP-Sil-8). In the enantioselective separation of PBBs in the eggs of the white-tailed sea eagle, it was not possible to establish the enantiomeric fraction with GC-ECNI-MS, whereas GC-EIMS/MS allowed for its verification (Götsch et al. 2005). When GC-ECNI-MS is combined with PTV injection (300°C isotherm), the limits of detection obtained are in the range of 1.5 ppb for PBB 153 (Loconto 2008). The GC-ECNI-MS method also generates a response for PBDEs. PBDEs are usually more abundant than PBBs; hence, a more selective method is needed for the determination of PBBs. The use of GC-ECNI-MS/MS resolves the problem of coelution between PBBs and PBDEs, and it is possible to determine the degree of bromination of the PBBs found in biological samples from fish and marine mammals (Vetter 2001; von der Recke and Vetter 2008a, b). It also allows for the recognition of artifacts and important information related to PBBs as compared with PBDEs (BDE 47 or BDE 154 versus PBB 153). The comprehensive two-dimensional GC (GCxGC, coupled to a μECD or a timeof-flight mass spectrometry, TOF-MS) has proved to be a powerful tool in the determination of different BFRs, their decomposition products, and other compounds coextracted from environmental samples (Korytár et al. 2005). While GCxGC guaranteed the chromatographic separation of the compounds, TOF-MS allowed for the mass spectral deconvolution of the coeluting compounds, as well as the use of 13C-labeled internal standards. This is not possible in GC-ECNI-MS. The flame ionization detector (FID) coupled to an LC-GC for the determination of PBBs in sediments (Kuosmanen et al. 2001) has limited application owing to its low sensitivity and/or selectivity.

339

The use of reversed- and normal-phase HPLC systems (Berger et al. 2002; von der Recke and Vetter 2007b, 2008a) and acetonitrile or acetonitrile/H2O (95 : 5, v/v) as an eluent is geared especially toward the selection or semipreparative enantioselective separations of PBBs (Gómara et al. 2006). Afterward, the resulting fractions may be determined by means of GC-MS/MS with an ion trap detector and PTV injection in samples of cheese, milk, and mackerel. Also, proton nuclear magnetic resonance (1H NMR) was used to verify the PBB structures (von der Recke and Vetter 2007b). A chemiluminesence method has been used to investigate the effects of PBBs on intracellular calcium and respiratory burst in human granulocytes, comparing their efficiency with the corresponding PCBs (Kristoffersen et al. 2002). Quality assurance (QA) comprises a group of procedures that includes quality control (QC), the activities carried out to verify the quality of the data obtained in the analysis of BFRs. As a general rule, 20%–25% of the analysis time should be dedicated to analysis quality. To ensure the sufficient quality of the data, a number of different steps must be taken during the validation of the analytical methods and in the quality control of the process (Covaci et al. 2003). Calibration standard solutions must have certain characteristics and be stored in a specific way in order to avoid losses of greater than 2% in a period of 6–9 months. Quantification procedures based on the standard addition method (the use of compounds marked with 13C is recommended to apply the isotope dilution method in MS) are aimed at compensating for the losses in the whole analytical procedure, along with the use of a syringe standard to compensate for fluctuations between injections. The analytical procedure must be specific to each compound and matrix being investigated. The analytical characteristics of the method are considered to be an internal quality

340

Analysis of Endocrine Disrupting Compounds in Food

control upon the determination of the following parameters: repeatability (the same operating conditions during a short time period), intermediate precision (variation in the lab), reproducibility (precision between laboratories), and accuracy (estimated through the use of certified reference materials. External quality control is generally determined through participation in interlaboratory tests that facilitate the assessment and evaluation of the efficiency of the method as a whole (Covaci et al. 2008b). Therefore, a study of PBBs in trout by means of HRGC-EI-HRMS, followed EPA 8290 QA/QC protocols (EPA 1994). The criteria for peak identification included acceptable retention times (±0.05 min), peak shape, and ion ratios (±15%). The limits of detection of PBB were found at 0.001–0.004 ng/g wet wt. Relative response factors (RRF), based on the response of the analytical standard, were used to determine the amount of native PBB compounds in addition to the recovery of standards and surrogates (60%–120%) (Luross et al. 2002). Following similar criteria using a GC-EI-HRMS system, a LOQ of 0.1 ng/g lipid, a percent relative standard deviation of 7.9%, and a mean recovery of 74% were obtained (Sjödin et al. 2004).

Occurrence and specific epidemiology studies Losses of PBB released into the environment at their places of manufacture may reach up to 51 kg/1000 kg of product (Di Carlo et al. 1978; UNEP 2006). These losses may be attributed to different causes (WHO 1994; ATSDR 2004; UNEP 2006): • Emission to the air. In 1977, the maximum amount of leaks into the atmosphere in the form of particles in the production areas was calculated to have reached 1.1 kg of PBB per 1000 kg produced. • Losses to wastewaters from the quenching and washing of PBBs as they were recov-

ered from the reaction mass. In 1977 losses of PBB through the sewers in their production area were calculated to be 4.6 μg/kg of product. • Losses into the soil. Soil samples taken in the packaging and loading zones at the Michigan Chemical Corp. contained PBBs in concentrations of 3500 and 2500 mg/kg, respectively. • Losses in solid form into legal or illegal disposal sites of electronic equipment (ewaste) owing to leaks, evaporation, runoff, and lixiviation. Loss of PBBs in the form of solid waste into disposal sites amounted to around 50 g/kg of product. It is expected that residual concentrations of PBBs will continue to be found for many years to come in the soil and streams near e-waste disposal and waste recycling areas where workers do not use appropriate protective equipment (Zhao et al. 2008). Most PBBs have been used mainly in the production of plastics whose estimated halflife is from 5 to 10 years. From the time that production ceases, it is assumed that the presence of these pollutants is due to their recycling and processing at disposal sites and incineration (ATSDR 2004; Zhao et al. 2008). During the decade after the production of PBBs stopped, concentrations were measured in a large number of samples of the biota in the areas surrounding the manufacturing centers of Michigan where the accident causing the subsequent contamination occurred. At the end of the 1980s, PBBs, in concentrations ranging between 15 and 15,000 μg/kg (lipid base), were detected in fish from Lake Huron (ATSDR 2004). The concentrations of several congeners of PBBs were determined in trout from lakes Huron, Superior, Erie, and Ontario (Luross et al. 2002). PBB 153 and PBB 101 were found in high concentrations ranging from 0.189 to 2083 μg/kg and from 0.042 to 0.633 μg/kg of fresh weight, respectively. Terrestrial and aquatic birds have served as indicators for controlling the levels and

Polybrominated Biphenyls

effects of persistent pollutants due to their high position in the food chain (Verreault et al. 2006; Vetter et al. 2008a; Luo et al. 2009). The concentrations of PBBs in the eggs of birds that prey on fishes (storm petrel, little gull, herring gull, and the red-breasted merganser) collected from 1975 to 1980 on the islands used for nesting in the northwest of Lake Michigan and Green Bay fluctuated between 0.02 and 0.25 mg/kg (Heinz et al. 1983, 1985). Substantial levels (3–140 ng/g lipid base) of PBB 153 have recently been found in species of aquatic birds that frequent waste recycling areas in China (Luo et al. 2009). Moreover, in a study of 52 samples of eggs of the peregrine falcon, both wild and in captivity, conducted in Sweden from 1987 to 1999, mean concentrations of 81–84 ng/g of PBB 153 (lipid base)were detected (Johansson et al. 2009). Studies carried out in recent years have demonstrated the potential of long-range transport (Scheringer et al. 2006; de Wit et al. 2006). The monitoring data gathered show that hexa-BB has been detected in biota simples in remote areas such as the Arctic. In these regions of the Arctic and North Atlantic, where the traditional diet includes large predators (the seal in Greenland and the pilot whale in the Faeroe Islands), exposure has not ceased. The level of PBBs in the fat of the pilot whale, which is consumed on these islands, can reach up to 17 μg/kg (lipid base), indicating the presence of hexa-BB in the diet (UNEP 2006). Studies on mollusks (mussels and oysters) from Scotland showed low levels of PBBs, around 0.01–0.1 μg/kg, suggesting that contamination occurs by long-distance maritime and atmospheric transport, as has been recorded in the tissue of Arctic polar bears (Fernandes et al. 2008). Owing to the lipophilic nature of PBBs, they are accumulated in human breast milk and tissue. Therefore, these samples are considered to be indicators of exposure to pollutants through food or in occupational settings. Exposure to hexa-BBs after the accident in

341

Michigan has been evaluated in human tissue, in breast milk, and in children (Thomas et al. 2001; Blanck et al. 2002). The Michigan Long-Term PBB Study conducted on 4000 people affected by the 1973 incident continues to investigate and evaluate the effects of PBBs on health (MDCH 2009). A study carried out in the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, estimated that the mean half-life of PBBs in the human body is 10.8 years (Rosen et al. 1995). Studies have been done on the population living near industrial zones that produce or use PBBs and on farmers in Washington state who were later examined as a control group in connection with the Michigan PBB studies (WHO 1994). In these preliminary studies, it was concluded that PBB concentrations do not show any noticeable decrease over time (Wolff et al. 1979). Later, different research papers reported the same trend for levels of PBBs in fat and human serum (Sherman 1991; Sjödin et al. 2003). Recently, there has been a proposal of a model that describes the effect of a number of different specific factors on the decrease in PBB serum levels in women in the Michigan study (Blanck et al. 2000a; Terrell et al. 2008). A retrospective epidemiological study has examined the intergenerational transfer of PBBs from mother to child in 140 cases and its association with breastfeeding models based on the concentrations of PBBs detected in serum and milk (Joseph et al. 2009). The breastfeeding period (≥5.5 months) was a significant variable associated with children having detectable serum concentrations of PBBs. It has been demonstrated that the intergenerational transfer of PBBs resembles that of other related lipophilic compounds such as PCBs and PBDEs. A 1988 study reported the contamination of human breast milk in Europe, on the basis of a study from North Rhine-Wesphalia, Germany (Krüger et al. 1988; WHO 1994). The milk samples contained concentrations

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Analysis of Endocrine Disrupting Compounds in Food

of between 0.002 and 28 μg/kg (lipid base) of the congeners ranging from penta-BB to octaBB. The most abundant component was PBB 153, followed by PBB 187 and PBB 182. The concentrations of PBB 153 in human and cow milk collected in the North Rhine-Wesphalia region were 1 μg/kg and 0.03 μg/kg (lipid base), respectively (WHO 1994). A recent study has focused on the differences found in the presence of PBBs in human breast milk samples of varying origin. PBB 153 was the most abundant in the Danish samples, whereas PBB 155 was the predominant component in the samples from Finland (Shen et al. 2008). A retrospective study conducted on 444 women and 899 children born between 1975 and 1997 has proved that there is a negative effect on newborn birth weight if the mother presented with high serum concentrations of PBBs (Givens et al. 2007). PBBs with a low bromine content are metabolized in hydroxylated derivatives that are preferably eliminated in the urine. Highly brominated PBBs are retained or excreted without any alteration in the feces (ATSDR 2004). Current human exposure to PBBs is considered to be very low, given that PBBs are no longer being produced or used (ATSDR 2004). In China, however, levels of PBBs (57.77 ng/g, dry wt) comparable to PBDE levels (29.64 ng/g, dry wt) have been found in hair samples taken from workers at recycling sites that process e-waste products (Zhao et al. 2008). This shows the facility with which these products are bioaccumulated in the environment and their biomagnification in the food chain, as they are able to enter the human body through the respiratory system, the skin, or by ingestion.

Regulatory status In Europe, the use of PBBs has been collectively prohibited from textiles that may come in contact with the skin since 1983

(European Commission 1983). In accordance with the CEFIC/European Brominated Flame Retardant Industry Panel and the European manufacturer, the production and use of PBBs ceased as of the year 2000 (OSPAR 2001). However, even before these compounds stopped being manufactured, PBBs had been less in demand as compared to the widespread use and production of PBDEs. During the 1990s, the demand for products treated with deca-BB was limited to the Benelux countries, France, and southern Europe, at a level of under 2000 tons per year (Lassen et al. 1999). The manufacture of hexa-BB ended in 1974 in the United States as a result of the incident in Michigan that occurred the year before, whereas the production of deca-BB ended in 1979. Canada officially banned PBBs in 2003, classifying them as toxic substances under the Canadian Environmental Protection Act (CEPA) 1999 (Canada Gazette 2003). Japan and Australia never manufactured PBBs and prohibited their importation in 1978 (WHO 1994). The Japan-Philippines Economic Partnership Agreement (JPEPA) considers PBBs to be substances whose trade must be strictly regulated pursuant to the Basel Agreement (JPEPA 2007). Hexa-BB is listed in annex A of the Protocol on Persistent Organic Pollutants (POPs) of the Convention on Long Range Transboundary Air Pollution (LRTAP). HexaBB, along with other PBBs, are also included in the Rotterdam Convention FAO/PNUMA (UNEP 2006). However, BFRs are not included in the Stockholm Convention on POPs. IARC has classified deca-BB, hexa-BB, and octa-BB, into Group 2B, possibly carcinogenic to humans (IARC 1987). NTP considers PBBs as reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity in experimental animals (NTP 2005). In 1995, the Voluntary Industry Commitment (VIC) was signed with the Organization for Economic Cooperation and Development

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(OECD) to regulate the production, handling, and disposal of BFRs, including PBBs. European directive 2002/95/CE on restricting the use of dangerous substances in electronic and electrical equipment initially mandated that PBBs must not be present in these products as of July 1, 2006 (European Commission 2003). However, a subsequent modification to the above directive exempts materials containing PBBs from the prohibition because, in practice, the elimination or replacement of these specific materials is not yet possible (European Commission 2005). Based on the feasibility of performing analyses to measure chemical compounds on a routine basis in accredited laboratories, production volumes, the appearance of chemical compounds in food and animal feed, and their persistence in the environment and toxicity, the inclusion of PBB 153 in a European program for the control of feeds and food (EFSA 2006) is recommended. Also required is selective analytical methodology that will allow for the clear differentiation between PBB 153 and PBDE 154, because these two compounds can coelute under certain analysis conditions. Moreover, there are new technical guidelines that provide instructions for the handling, treatment, and rational disposal of PBB wastes in the environment (European Commission 2009). OSPAR aims to prevent the contamination of the maritime zone by reducing the dumping, release, and spillage of hazardous substances, achieving the end of this practice by the year 2020. With regard to food safety in December 2008 ATSDR defined a minimal risk level (MRL) of 0.01 mg/kg/day for acute-duration oral exposure to PBBs (ATSDR 2008).

Trends in PBBs analysis in food Below is a summary of some of the general conclusions and most important trends related to the state-of-the-art analysis of PBB wastes

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in food samples and their presence in the environment: • Despite all the prohibition measures adopted in recent years, the presence of PBBs has been demonstrated even in samples from regions with no apparent industrial load by virtue of long-range transport. These substances also possess mechanisms of action that have yet to be explained. Their lipophilic nature justifies their presence in the food chain, mainly in lipid-rich foods of animal origin such as meat, fish, and dairy products, which are part of the daily diet. Moreover, owing to their widespread use and the possible existence of their illegal production in developing nations and countries with economies in transition, it is to be expected that workers involved in processing wastes will present higher levels of some PBBs in the blood or other tissues, where these compounds can be bioaccumlated. • Analyte extraction efficiency is of key importance for the preparation of samples in food residue determination. There is a real need for new procedures that can reduce solvent consumption, costs, and error sources. Also important is the automation of the extraction and cleanup steps to allow on-line sample preparation and increase the robustness of the analytical methodology. • The hyphenation of GC/LC to different mass analyzers should increase the ability to obtain structural information and identify unknown compounds related to these analytes. Certified reference materials (CRMs) related to food matrices should be available for PBB determination in order to ensure the precision and accuracy of the analytical method. Also, there is a clear need for the validation of new techniques and procedures with a view toward their adoption as official methods (e.g., AOAC protocols), as substitutes for the more laborious, timeconsuming, and classical procedures.

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Acknowledgments The authors wish to thank the Galician Government, Xunta de Galicia, for financial support (Project No. PGIDIT06PXIB 237039PR).

References Agency for Toxic Substances and Disease Registry (ATSDR). 2004. Toxicological profile for polybrominated biphenyls and polybrominated diphenyl ethers. U.S. Department of Health and Human Services. Public Health Service. Agency for Toxic Substances and Disease Registry. Available at: http:// www.atsdr.cdc.gov/toxprofiles/tp68.html. Agency for Toxic Substances and Disease Registry (ATSDR). 2008. Available at: http://www.atsdr.cdc. gov/mrls/pdfs/atsdr_mrls_december_2008.pdf. Alaee M., Arias P., Sjödin A., Bergman Å. 2003. An overview of commercially used brominated flame retardants, their applications, their use patterns in different countries/regions and possible modes of release. Environ. Int. 29:683–689. Anderson H.A. 1985. Utilization of adipose tissue biopsy in characterizing human halogenated hydrocarbon exposure. Environ. Health Perspect. 60:127–131. Bailey R.A, Clark H.M., Ferris J.P., Krause S., Strong R.L. 2002. Chemistry of the Environment, 2nd ed. Oxford, Elsevier. Berger U., Vetter W., Götsch A., Kallenborn R. 2002. Chromatographic enrichment and enantiomer separation of axially chiral polybrominated biphenyls in a technical mixture. J. Chromatogr. A. 973:123–133. Blanck H.M., Marcus M., Hertzberg V., Tolbert P.E., Rubin C., Henderson A.K., Zhang R.H. 2000a. Determinants of polybrominated biphenyl serum decay among women in the Michigan PBB cohort. Environ. Health Perspect. 108:147–152. Blanck H.M., Marcus M., Tolbert P.E., Rubin C., Henderson A.K., Hertzberg V.S., Zhang R.H., Cameron L. 2000b. Age at menarche and tanner stage in girls exposed in utero and postnatally to polybrominated biphenyl. Epidemiology. 11:641–647. Blanck H.M., Marcus M., Rubin C., Tolbert P.E., Hertzberg V.S., Henderson A.K., Zhang R.H. 2002. Growth in girls exposed in utero and postnatally to polybrominated biphenyls and polychlorinated biphenyls. Epidemiology. 13:205–210. Bordajandi L.R., Gómez G., Fernández M.A., Abad E., Rivera J., González M.J. 2003. Study on PCBs, PCDD/Fs, organochlorine pesticides, heavy metals and arsenic content in freshwater fish species from the River Turia (Spain). Chemosphere. 53:163–171. Braune B.M., Mallory M.L., Gilchrist H.G., Letcher R.J., Drouillard K.G. 2007. Levels and trends of organochlorines and brominated flame retardants in Ivory Gull eggs from the Canadian Arctic, 1976 to 2004. Sci. Total Environ. 378:403–417.

Brinkman U.A.T., de Kok A. 1980. Halogenated biphenyls, terphenyls, naphthalenes, dibenzodioxins, and related products. Production, properties, and usage. Top Environ Health. 4:1–40. Brown G.G., Preisman R.C., Anderson M.D., Nixon R.K., Isbister J.L., Price H.A. 1981. Memory performance of chemical workers exposed to polybrominated biphenyls. Science. 212:1413–1415. Brown D.J., Van Overmeire I., Goeyens L., Denison M.S., De Vito M.J., Clark G.C. 2004. Analysis of Ah receptor pathway activation by brominated flame retardants. Chemosphere. 55:1509–1518. Canada Gazette. 2003. Prohibition of certain toxic substances regulations 137. April 9. Available at: http://www.gazette.gc.ca/archives/p2/2003/2003-0409/html/sor-dors100-eng.html. Capen C.C. 1992. Pathophysiology of chemical injury of the thyroid-gland. Toxicol. Lett. 64:381–388. Carro A.M., Lorenzo R.A., Fernández F., Rodil R., Cela R. 2005. Multi-residue screening of chlorinated and brominated compounds from aquaculture samples using matrix solid-phase dispersion—gas chromatography–mass spectrometry. J. Chromatogr. A. 1071:93–98. Carro A.M., Lorenzo R.A., Fernández F., Rodil R., PhanTan-Luu R., Cela R. 2007. Microwave-assisted extraction followed by headspace solid-phase microextraction and gas chromatography with mass spectrometry detection (MAE-HSSPME-GC-MS/MS) for determination of polybrominated compounds in aquaculture samples. Anal. Bioanal. Chem. 388:1021–1029. Chhabra R.S., Bucher J.R., Haseman J.K., Elwell M.R., Kurtz P.J., Carlton B.D. 1993. Comparative carcinogenicity of polybrominated biphenyls with or without perinatal exposure in rats and mice. Fundam. Appl. Toxicol. 21:451–460. Covaci A., Voorspoels S., de Boer J. 2003. Determination of brominated flame retardants, with emphasis on polybrominated diphenyl ethers (PBDEs) in environmental and human samples—a review. Environ. Int. 29:735–756. Covaci A., Voorspoels S., D’Silva K., Huwe J., Harrad S. 2008a. Brominated flame retardants as food contaminants. In: Picó Y., ed., Comprehensive Analytical Chemistry, vol. 51. Elsevier B.V., pp. 507–570. Covaci A., Voorspoels S., Ramos L., Neels H., Blust R. 2007. Recent developments in the analysis of brominated flame retardants and brominated natural compounds. J. Chromatogr. A. 1153:145–171. Covaci A., Dirtu A.C. 2008b. Brominated flame retardants: Analytical, toxicological, and environmental aspects. In: Popescu, C., Zamfir, A.D., Dinca, N., eds., Applications of Mass Spectrometry in Life Safety. Springer Science, pp. 153–184. Darnerud P.O. 2008. Bromianted flame retardants as possible endocrine disrupter. Int J. Androl. 31: 152–160. de Boer J., Wester P.G., Rodriquez D.P., Lewis W.E., Boon J.P. 1998. Polybrominated biphenyls and diphenylethers in sperm whales and other marine mammals. A new threat to ocean life? Organohal. Comp. 35:383–386.

Polybrominated Biphenyls

de Boer J., de Boer K., Boon J.P. 2000. Polybrominated biphenyls and diphenylethers. In: Paasivirta, J., ed., The Handbook of Environmental Chemistry, vol. 3, part K. Springer Verlag, Berlin, pp. 61–95. de Boer J., Allchin C., Law R., Zegers B., Booij J.P. 2001. Method for the analysis of polybrominated diphenylethers in sediments and biota. Trends Anal. Chem. 20:591–599. de Wit C.A. 2002. An overview of brominated flame retardants in the environment. Chemosphere. 46:583–624. de Wit C.A., Alaee M., Muir D.C.G. 2006. Levels and trends of brominated flame retardants in the Arctic. Chemosphere. 64:209–233. di Carlo F.J., Seifter J., DeCarlo V.J. 1978. Assessment of the hazards of polybrominated biphenyls. Environ. Health Perspect. 23:351–365. D’Silva K., Fernández A., Rose M. 2004. Brominated organic micropollutants: Igniting the flame retardant issue. Crit. Rev. Environ. Int. 34:141–207. Dunckel A.E. 1975. An updating on the polybrominated biphenyl disaster in Michigan. J. Amer. Vet. Med. Assoc. 167:838–841. EPA 2007. http://www.epa.gov/osw/hazard/testmethods/ sw846/pdfs/3535a.pdf Environmental Protection Agency (EPA). 1994. Method 8290. Available at: http://www.caslab.com/EPAMethods/PDF/8290.pdf. European Commission (EC). 1983. Directive 83/264/EC of 16 May 1983 amendment for the fourth time Directive 76/769/EEC on the approximation of the laws, regulations, and administrative provisions of the Member States relating to restrictions on the marketing and use of certain dangerous substances and preparations. Off. J. Eur. Commun. L, 147:9–10. European Commission (EC). 2003. Directive 2002/95/ EC of the European Parliament and of the Council of 27 January 2003 on the restriction of the use of certain hazardous substances in electrical and electronic equipment. Off. J. Eur. Commun. L 37, 46:19–23. European Commission (EC). 2005. Directive 2005/717/ EC: Commission Decision of 13 October 2005 amending for the purposes of adapting to the technical progress the Annex to Directive 2002/95/EC of the European Parliament and of the Council on the restriction of the use of certain hazardous substances in electrical and electronic equipment. Off. J. Eur. Commun. L 271, 48:48–50. European Commission (EC). 2009. Directive 2009/C 175/06: Opinion of the European Economic and Social Committee on ‘Meeting the challenges of the WEEE management in the EU’. Off. J. Eur. Commun. C 175, 52:34–36. European Food Safety Authority (EFSA). 2006. Advice of the scientific panel on contaminants in the food chain on a request from the commission related to relevant chemical compounds in the group of brominated flame retardants for monitoring in feed and food. (Question N° EFSA-Q-2005–244). EFSA J. 328:1–4. Evenset A., Christensen G.N., Kallenborn R. 2005. Selected chlorobornanes, polychlorinated naphthalenes, and brominated flame retardants in Bjørnøya

345

(Bear Island) freshwater biota. Environ. Pollut. 136:419–430. Fajar N.M., Carro A.M., Lorenzo R.A., Fernandez F., Cela R. 2008. Optimization of microwave-assisted extraction with saponification (MAES) for determination of polybrominated flame retardants in aquaculture samples. Food Add. Contamin. 25:1015–1023. Fernandes A., White S., D’Silva K., Rose M. 2004. Simultaneous determination of PCDDs, PCDFs, PCBs, and PBDEs in food. Talanta. 63:1147–1155. Fernandes A., Dicks P., Mortimer D., Gem M., Smith F., Driffield M., White S., Rose M. 2008. Brominated and chlorinated dioxins, PCBs, and brominated flame retardants in Scottish shellfish: Methodology, occurrence and human dietary exposure. Mol. Nutr. Food Res. 52:238–249. Fernandes A., Mortimer D., Gem M., Dicks P., Smith F., White S., Rose M. 2009. Brominated dioxins (PBDDs/ Fs) and PBDEs in marine shellfish in the UK. Food Addit. and Contamn. 26:918–927. Focant J.F., Sjödin A., Turner W.E., Patterson D.G., Jr. 2004. Measurement of select polybrominated diphenyl ethers, polybrominated and polychlorinated biphenyls, and organochlorine pesticides in human serum and milk using comprehensible two-dimensional gas chromatography isotope dilution time-of-flight mass spectrometry. Anal. Chem. 76:6313–6320. Gao F., Luo X., Yang Z., Wang X., Mai B. 2009. Brominated flame retardants, polychlorinated biphenyls, and organochlorine pesticides in bird eggs from the Yellow River Delta, North China. Environ. Sci. Technol. 43:6956–6962. García-López M., Canosa P., Rodríguez I. 2008. Trends and recent applications of matrix solid-phase dispersion. Anal. Bioanal. Chem. 391:963–974. Georlette, P. 2009. Environmental benefits of brominated fire retardants. Paper read at FRT09: Fire Retardant Technologies 2009, 21–23 April, at UCLan (University of Central Lancashire), Preston, UK. Getty S.M., Rickert D.E., Trapp A.L., Buck W.B. 1977. Polybrominated biphenyl (PBB) toxicosis: An environmental accident. Crit. Rev. Environ. Control. 7:309–324. Givens M.L., Small C.M., Terrell M.L., Cameron L.L., Blanck H.M., Tolbert P.E., Rubin C., Henderson A.K., Marcus M. 2007. Maternal exposure to polybrominated and polychlorinated biphenyls: Infant birth weight and gestational age. Chemosphere. 69:1295–1304. Gómara B., García-Ruiz C., González M.J., Marina M.L. 2006. Fractionation of chlorinated and brominated persistent organic pollutants in several food samples by pyrenyl-silica liquid chromatography prior to GC–MS determination. Anal. Chim. Acta. 565: 208–213. Götsch A., Mariussen E., von der Recke R., Herzke D., Berger U., Vetter W. 2005. Analytical strategies for successful enantioselective separation of atropisomeric polybrominated biphenyls 132 and 149 in environmental samples. J. Chromatogr. A. 1063: 193–199. Hardy M.L. 2002. A comparison of the properties of the major commercial PBDPO/PBDE product to those

346

Analysis of Endocrine Disrupting Compounds in Food

of major PBB and PCB products. Chemosphere. 46:717–728. Heinz G.H., Haseltine S.D., Reichel W.L., Hensler G.L. 1983. Relationships of environmental contaminants to reproductive success in red-breasted mergansers (Mergu serrator) from Lake Michigan. Environ. Pollut. Ser. A 32:211–232. Heinz G.H., Erdman T.C., Haseltine S.D., Stafford C. 1985. Contaminant levels in colonial waterbirds from Green Bay and Lake Michigan, 1975–80. Environ. Monit. Assess. 5:223–236. Henck J.W., Mattsson J.L., Rezabek D.H., Carlson C.L., Rech R.H. 1994. Developmental neurotoxicity of polybrominated biphenyls. Neurotoxicol. Teratol. 16:391–399. Henderson A.K., Rosen D., Miller G.L., Figgs L.W., Zahm S.H., Sieber S.M., Humphrey H.E.B., Sinks T. 1995. Breast cancer among women exposed to polybrominated biphenyls. Epidemiology. 6:544–546. Herzke D., Kallenborn R., Nygard T. 2002. Organochlorines in egg samples from Norwegian birds of prey: Congener-, isomer- and enantiomer specific considerations. Sci. Total Environ. 291:59–71. Herzke D., Kallenborn R., Nygård T., Vetter W. 2005. Brominated flame retardants and other organobromines in Norwegian predatory bird eggs. Chemosphere. 61:441–449. Hill R.H., Patterson Jr. D.G., Orti D.L., Holler J.S., Needham L.L., Sirmans S.L., Liddle J.A. 1982. Evidence of degradation of polybrominated biphenyls in soil samples from Michigan. J. Environ. Sci. Health B. 17:19–33. Hites R.A. 2006. The Handbook of Environmental Chemistry, vol. 5, part N. Springer Verlag, Heidelberg. Hoque A., Sigurdson A.J., Burau K.D., Humphrey H.E., Hess K.R., Sweeney A.M. 1998. Cancer among a Michigan cohort exposed to polybrominated biphenyls in 1973. Epidemiology. 9:373–378. International Agency for Research on Cancer (IARC). 1986. Some halogenated hydrocarbons and pesticide exposures. Summary of data reported and evaluation. Monographs on the Evaluation of Carcinogenic Risks to Humans. 41:261–292. Available at: http://monographs.iarc.fr/ENG/Monographs/vol41/ volume41.pdf. International Agency for Research on Cancer (IARC). 1987. Overall evaluations of carcinogenicity: An Updating of IARC Monographs. Monographs on the Evaluation of Carcinogenic Risks to Humans. 1–42 Suppl, 7:321. Available at: http://monographs.iarc.fr/ ENG/Monographs/suppl7/suppl7.pdf. Jansson B., Andersson R., Asplund L., Litzen K., Nylund K., Sellstrom U., Uvemo U.B., Wahlberg C., Wideqvist U. 1993. Chlorinated and brominated persistent organic compounds in biological samples from the environment. Environ. Toxicol. Chem. 12:1163–1174. Jaspers V.L.B., Covaci A., Voorspoels S., Dauwe T., Eens M., Schepens P. 2006. Brominated flame retardants and organochlorine pollutants in aquatic and terrestrial predatory birds of Belgium: Levels, patterns, tissue distribution, and condition factors. Environ. Pollut. 139:340–352.

Johansson A.-K., Sellström U., Lindberg P., Bignert A., de Wit C.A. 2009. Polybrominated diphenyl ether congener patterns, hexabromocyclododecane, and brominated biphenyl 153 in eggs of peregrine falcons (Falco peregrinus) breeding in Sweden. Environ. Tox. Chem. 28:9–17. Joseph A.D., Terrell M.L., Small C.M., Cameron L.L., Marcus M. 2009. Assessing inter-generational transfer of a brominated flame retardant. J. Environ. Monit. 11:802–807. JPEPA as a Step in Japan’s Greater Plan to Liberalize Hazardous Waste Trade in Asia. 2007. Basel Action Network. A Project of Earth Economics. 29 August. Available at: http://www.ban.org/Library/ JPEPA_Report_BAN_FINAL_29_Aug_071.pdf. Kay K. 1977. Polybrominated biphenyls (PBB) environmental contamination in Michigan, 1973–1976. Environ. Res. 13:74–93. Kimbrough R.D. 1987. Human health effects of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). Annu. Rev. Pharmacol. Toxicol. 27:87–111. Korytár P., Covaci A., Leonards P.E.G., de Boer J., Brinkman U.A.Th. 2005. Comprehensive twodimensional gas chromatography of polybrominated diphenyl ethers. J. Chromatogr. A. 1100:200–207. Kristoffersen A., Voie Ø.A., Fonnum F. 2002. Orthosubstituted polybrominated biphenyls activate respiratory burst in granulocytes from humans. Toxicol. Lett. 129:161–166. Krüger C., Fürst P., Gröbel W. 1988. Detection and determination of polybrominated biphenyls in human milk. Dtsch Lebensm Rundsch. 84: 273–276 (in German). Kuosmanen K., Hyötyläinen T., Hartonen K., Riekkola M.-L. 2001. Pressurised hot water extraction coupled on-line with liquid chromatography–gas chromatography for the determination of brominated flame retardants in sediment samples. J.Chromatogr. A. 943:113–122. Lambert G.H., Hsu C.C., Humprey H., Chen J., Schoeller D., Mortensen K. 1992. Cytochrome P4501A2 in vivo induction—a potential biomarker of polyhalogenated biphenyls and their related chemicals effects on the human. Chemosphere. 25:197–200. Lassen C., Løkke S., Andersen L.I. 1999. Brominated flame retardants—substance flow analysis and assessment of alternatives. Project No. 494. Danish Environmental Agency, Ministry of Environment and Energy, Copenhagen, Denmark. Loconto P.R. 2008. Biomonitoring: Polybrominated diphenyl ethers and polybrominated biphenyls using capillary GC with electron-capture negative ion mass selective detection. LC-GC North America. 26:1118–1130. Luo X.J., Zhang X.L., Liu J., Wu J.-P., Luo Y., Chen S.J., Mai B.-X., Yang Z.-Y. 2009. Persistent halogenated compounds in waterbirds from an e-waste recycling region in south China. Environ. Sci. Technol. 43:306–311. Luross J.M., Alaee M., Sergeant D.B., Cannon C.M., Whittle D.M., Solomon K., Muir D.C.G. 2002. Spatial distribution of polybrominated diphenyl ethers and

Polybrominated Biphenyls

polybrominated biphenyls in lake trout from the Laurentian Great Lakes. Chemosphere. 46:665–672. Mendiola J.A., Herrero M., Cifuentes A., Ibañez E. 2007. Use of compressed fluids for sample preparation food application. J. Chromatogr. A. 1152:234–246. Miceli J.N., Nolan D.C., Marks B., Hariharan M. 1985. Persistence of polybrominated biphenyls (PBB) in human post-mortem tissue. Environ. Health Perspect. 60:399–403. Michigan Department of Community Health (MDCH). 2009. Available at: http://www.michigan.gov/mdch. Morris P.J., Quensen J.F., Tiedje J.M., Boyd S.A. 1992. Reproductive debromination of the commercial polybrominated biphenyl mixture Firemaster BP6 by anaerobic microorganisms from sediments. Appl. Environ. Microbiol. 58:3249–3256. National Toxicology Program (NTP). 1992. NTP technical report on the perinatal toxicology and carcinogenesis studies of polybrominated biphenyls (Firemaster FF-1) (CAS No. 67774-32-7) in F344/N rats and B6C3F1 mice (feed studies). National Toxicology Program, Research Triangle Park, N.C. National Toxicology Program (NTP). 2005. Report on Carcinogens, 11th ed., Public Health Service, U.S. Department of Health and Human Services. Available at:http://ntp.niehs.nih.gov/index.cfm?objectid=32BA9724F1F6-975E-7FCE50709CB4C932. OSPAR Commission. 2001 (2004 update). Certain Brominated Flame Retardants—Polybrominated Diphenylethers, Polybrominated Biphenyls, Hexabromo Cyclododecane. Available at: http:// www.ospar.org/documents/dbase/publications/ p00135_BD%20on%20Brominated%20flame%20 retardants.pdf. Polo M., Gómez-Noya G., Quintana J.B., Llompart M., García Jares C., Cela R. 2004. Development of a solidphase microextraction gas chromatography/tandem mass spectrometry method for polybrominated diphenylethers and polybrominated biphenyls in water samples. Anal. Chem. 76:1054–1062. POPRC. Persistent Organic Pollutants Review Committee of the Stockholm Convention. 2007. Hexabromobiphenyl draft risk management evaluation. Third meeting of POPRC. Geneva, Switzerland. Prieto A., Zuloaga O., Usobiaga A., Etchebarria N., Fernández L.A. 2008. Use of experimental design in the optimisation of stir bar sorptive extraction followed by thermal desorption for the determination of brominated flame retardants in water samples. Anal. Bioanal. Chem. 390:739–748. Programa de las Naciones Unidas para el Medio Ambiente (PNUMA). 1992. Aplicacion del procedimiento de información y consentimiento previos a las sustancias químicas prohibidas o severamente limitadas que circulan en el comercio internacional. PROGRAMA CONJUNTO FAO/PNUMA. Roma– Ginebra. Rangga-Tabbu C., Sleight S.D. 1992. Development of preneoplastic lesions in the liver and nasal epithelium of rats initiated with N-nitrosodimethylamine or N-nitrosopyrrolidine and promoted with polybrominated biphenyls. Food Chem. Toxicol. 30:921– 926.

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Rodil R., Carro A.M., Lorenzo R.A., Cela R. 2005a. Selective extraction of levels of polychlorinated and polybrominated contaminants by supercritrical fluidsolid-phase microextraction and determination by gas chromatography mass/spectrometry. Application to aquaculture fish, feed, and cultured marine species. Anal. Chem. 77:2259–2265. Rodil M.R. 2005b. Desarrollo de nuevas estrategias de preparación de muestras aplicadas al control de contaminantes medioambientales a niveles traza. Ph.D. Thesis, Universidad de Santiago de Compostela, Spain. Rodil R., Carro A.M., Lorenzo R.A., Cela R. 2007. Multicriteria optimisation of a simultaneous supercritical-fluid-extraction and clean-up procedure for the determination of persistent organohalogenated pollutants in aquaculture samples. Chemosphere. 67:1453–1462. Rosen D.H., Flanders W.D., Friede A., Humphrey H.E.B., Sinks T.H. 1995. Half-life of a polybrominated biphenyl in human sera. Environ. Health Perspect. 103:272–274. Salgado-Petinal C., Garcia-Chao M, Liompart M., Garcia-Jares C., and Cela R. 2006a. Headspace solidphase microextraction gas chromatography tandem mass spectrometry for the determination of brominated flame retardants in environmental solid samples. 385(3):637–644. Scheringer M., MacLeod M., Wegmann F. 2006. Analysis of four current POP candidates with the OECD Pov and LRTP screening tool. Available at: http://www.sustchem.ethz.ch/downloads/. SciFinder Scholar web version. 2009. American Chemical Society. http://www.chem.ox.ac.uk/cheminfo/ scifinder.html. Shen H., Main K.M., Andersson A.-M., Damgaard I.N., Virtanen H.E., Skakkebaek N.E., Toppari J., Schramm K.-W. 2008. Concentrations of persistent organochlorine compounds in human milk and placenta are higher in Denmark than in Finland. Human Reprod. 23:201–210. Sherman JD. 1991. Polybrominated biphenyl exposure and human cancer: Report of a case and public health implications. Toxicol. Ind. Health 7:197–205. Sjödin A., Jones R.S., Lapeza C., Focant J.-F., Wang R., Turner W.E., Needham L.L., Patterson Jr. D.G. 2003. Retrospective time trend study of brominated flame retardants and polychlorinated biphenyls in human serum from various regions of the United States, 1985–2002. Organohalogen Compounds. 61:1–4. Sjödin A., McGahee III E.E., Focant J.-F., Jones R.S., Lapeza C.R., Zhang Y., Patterson Jr. D.G. 2004. Semiautomated high-throughput extraction and cleanup method for the measurement of polybrominated diphenyl ethers and polybrominated and polychlorinated biphenyls in breast milk. Anal. Chem. 76:4508–4514. Stapleton H.M. 2006. Instrumental methods and challenges in quantifying polybrominated diphenyl ethers in environmental extracts: A review. Anal. Bioanal. Chem. 386:807–817. Stepniczka H. 1976. Complete bromination of nonfused ring aromatic compounds. US Patent No. US 3965197 Appl. No. US 1972-222412.

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Analysis of Endocrine Disrupting Compounds in Food

Terrell M.L., Manatunga A.K., Small C.M., Cameron L.L., Wirth J., Blanck H.M., Lyles R.H., Marcus M. 2008. A decay model for assessing polybrominated biphenyl exposure among women in the Michigan long-term PBB study. J. Exposure Sci. Environ. Epidemiol. 18:410–420. Thomas A.R., Marcus M., Zhang R.H., Blanck H.M., Tolbert P.E., Hertzberg V., Henderson A.K., Rubin C. 2001. Breast-feeding among women exposed to polybrominated biphenyls in Michigan. Environ. Health Perspect. 109:1133–1137. United Nations Environment Program (UNEP). 2006. UNEP/POPS/POPRC.2/9: Draft Risk Profile: Hexabromobiphenyl. In: Stockholm Convention on Persistent Organic Pollutants (POPs). UNEP, Geneva, Switzerland. van Leeuwen S.P.J., de Boer J. 2008. Brominated flame retardants in fish and shellfish-levels and contribution of fish consumption to dietary exposure of Dutch citizens to HBCD. Mol. Nutr. Food Res. 52:194–203. Verreault J., Villa R.A., Gabrielsen G.W., Skaare J.U., Letcher R.J. 2006. Maternal transfer of organohalogen contaminants and metabolites to eggs of Articbreeding glaucous gulls. Environ. Poll. 144: 1053–1060. Vetter W. 2001. A GC/ECNI-MS method for the identification of lipophilic anthropogenic and natural brominated compounds in marine samples. Anal. Chem. 73:4951–4957. Vetter W., von der Recke R., Herzke D., Nygård T. 2008a. Detailed analysis of polybrominated biphenyl congeners in bird eggs from Norway. Environ. Poll. 156:1204–1210. Vetter W., Von der Recke R., Symons R. 2008b. Determination of polybrominated biphenyls in Tasmanian devils (Sarcophilus harrisii) by gas chromatography coupled to electron capture negative ion tandem mass spectrometry or electron ionization highresolution mass spectrometry. Rapid Commun. Mass Spectrom. 22:4165–4170. von der Recke R., Vetter W. 2007a. GC-ECNI-MSMS residue pattern of hexabrominated biphenyls in marine

mammals and fish. Organohalogen Compounds. 69:505/1–505/4. von der Recke R., Vetter W. 2007b. Photolytic transformation of polybrominated biphenyls leading to the structures of unknown hexa- to nonabromo-congeners. J. Chromatogr. A. 1167:184–194. von der Recke R., Vetter W. 2008a. Anaerobic transformation of polybrominated biphenyls with the goal of identifying unknown hexabromobiphenyls in Baltic cod liver. Chemosphere. 71:352–359. von der Recke R., Vetter W. 2008b.Congener pattern of hexabromobiphenyls in marine biota from different provenientes. Science Tot. Environm. 393:358–366. Wang R.Y., Needham L.L. 2007. Environmental chemicals: From the environment to food, to breast milk, to the infant. J. Toxicol. Environ. Health, Part B. 10:597–609. Wolff M.S., Anderson H.A., Rosenman K.D., Selikoff I.J. 1979. Equilibrium of polybrominated biphenyl (PBB) residues in serum and fat of Michigan residents. Bull. Environ. Contam. Toxicol. 21:775–781. World Health Organization (WHO). 1994. Environmental Health Criteria 152: Polybrominated biphenyls. International Program on Chemical Safety, World Health Organization, Geneva, Switzerland. Available at: http://www.inchem.org/documents/ehc/ehc/ehc152. htm. Zhao G., Wang Z., Dong M.H., Rao K., Luo J., Wang D., Zha J., Huang S., Xu Y., Ma M. 2008. PBBs, PBDEs and PCBs levels in hair of residents around e-waste disassembly sites in Zhejiang Province, China, and their potential sources. Sci. Total Environ. 397:46–57. Zhao G., Zhou H., Wang D., Zha J., Xu Y., Rao K., Ma M., Huang S., Wang Z. 2009. PBBs, PBDEs, and PCBs in food collected from e-waste disassembly sites and daily intake by local residents. Sci. Total Environ. 407:2565–2575. Zhao H.X., Zhang Q., Xue X.Y., Liang X.M., Kettrup A. 2005. Predicting gas chromatographic retention times of 209 polybrominated diphenyls (PBBs) for different temperature programs. Anal. Bioanal. Chem. 382: 1304–1310.

Chapter 15 Bisphenol A Ana Ballesteros-Gómez and Soledad Rubio

Introduction Bisphenol A (4-4′-isopropylidenediphenol or BPA; CAS: 80-05-7) is a recognized endocrine disruptor (Lyons 2000; Chen et al. 2002) and one of the chemicals produced in high volumes in the world (Burridge 2003). Its chemical structure consists of two phenol rings connected by a methyl bridge (see Figure 15.1). The BPA market had an annual global capacity of 3.9 million tons in 2006, and it was forecasted to grow up near 5 million tons in 2010 (Morgan 2006). BPA is used mainly as building blocks in the manufacture of epoxy resins and polycarbonate plastic. Other secondary outlets include flame retardants (mainly tetrabromobisphenol-A), unsaturated polyester resins, and polyacrylate, polyetherimide, and polysulfone resins. These BPA-based polymers have a wide array of uses as food contact materials; namely, epoxy resins are used as protective coatings in a variety of food containers and polycarbonate plastic is used to make infant feeding bottles, microwave ovenware, storage containers, reusable bottles, and water pipes, among others. The estrogenic activity of BPA was first reported in 1993 (Krishnan et al. 1993) and it was classified as a very weak endocrine disruptor. Its affinity for estrogen receptors is

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

10,000- to 100,000-fold weaker than that of estradiol. Currently, the tolerable daily intake (TDI) set by the European Union (EU) Commission (EFSA 2006) and the reference dose (RfD) established by EPA (IRIS 1988) is 0.05 mg BPA/kg body weight/day. This value was calculated on the basis of a 100fold uncertainty factor to the accepted nonobserved-adverse-effect level (NOAEL) of 5 mg/kg. On the other hand, a specific migration limit (SML) for BPA from food contact with plastic materials of 600 ng g−1 was set by the EU Commission in 2004 (EC 2004). An extensive body of new literature concerning low-dose effects of BPA has been published on the basis of new genomic and nongenomic estrogen-response mechanisms, with the disruption of the cell function occurring at doses of 1 pM (0.23 ng L−1) (reviewed in Vom Saal and Hughes 2005). Recent studies have also shown the potential of BPA to disrupt thyroid hormone action (Zoeller et al. 2005), to cause proliferation of human prostate cancer cells (Wetherill et al. 2002), and to block testosterone synthesis (Akingbemi et al. 2004) at parts per trillion levels. These recent findings have given rise to controversy about the BPA limit values set by regulatory agencies for consumer health protection, thus a new risk assessment has been strongly recommended (Vom Saal and Hughes 2005). Because of the high volume, wide dispersive use, and endocrine-disrupting and toxic properties of BPA, reliable analytical methods are needed for risk assessment and control of human exposure. Basic requirements for 349

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these methods are high selectivity and sensitivity due to the complexity and variety of matrices used and the low levels of BPA in food (see Table 15.1). As a result, extensive sample treatment is usually needed before quantitation of BPA. Figure 15.2 shows the most common steps required for BPA determination, including removal of background

contamination, sample preparation, extraction, cleanup, and instrumental analysis. Here, the state of the art of the analytical methodologies developed to determine BPA in food are presented and critically discussed in terms of simplicity, robustness, cost, sensitivity, and selectivity. Main drawbacks and the need for future developments are highlighted.

Removal of background contamination Laboratory contamination of BPA can occur easily due to the ubiquity of BPA-based polymers. Contamination mainly arises from

Figure 15.1. Bisphenol A chemical structure.

REMOVAL OF BACKGROUND CONTAMINATION

Hea t- tre a ted g las s war e

So l ve nt - w a sh e d ma t eri al SAMPLE PREPARATION

Homogeneization

Degassing (beverages)

Filtration

Freeze-drying

EXTRACTION SOLVENT-BASED

SORBENT-BASED SPE, SPME SBSE, MSPD

LLE, SE, MAE, PLE, SSE CLEAN-UP

PROTEINS (acid and/or organic solvent addition SPE (Oasis HLB, Florisil C18) followed by ultracentrifugation) IAC GENERAL

,

LIPIDS (apolar solvent or freezing lipid filtration)

INSTRUMENTAL ANALYSIS

(Derivatization)

LC

MS MS-MS

FL

ED

IMMUNOCHEMICAL METHODS

GC-MS

Figure 15.2. Analytical methodologies for the determination of bisphenol A in foodstuffs. LLE, liquid–liquid extraction; SE, solvent extraction; MAE, microwave-assisted extraction; PLE, pressurized liquid extraction; SSE, supramolecular solvent extraction; SPE, solid-phase extraction; SPME, solid-phase microextraction; SBSE, stir bar sorptive extraction; MSPD, matrix solid-phase dispersion; FL, fluorescence detection; ED, electrochemical detection.

Table 15.1. Levels of BPA found in foods. Foodstuff (Country of origin) Fruits and vegetables Japan, China, USA UK, Italy, Belgium, Canada New Zealand, Thailand, Alaska, Italy, South Africa, USA Germany, Italy, Thailand, Indonesia, South Africa Spain

BPA Concentration

18.4–95 ng g−1 9–48 ng g−1 12–24 ng g−1

References

14(8) 10(10) 33(11)

a

10(10)

5–35 ng g−1

Yoshida et al. 2001 Goodson et al. 2002 Thomson and Grounds 2005 Braunrath et al. 2005b

80–420 ng g−1

García-Prieto et al. 2008

6(6)

a

Milk — — China

8(2) 10(3) 8(4)

7.11–15.2 ng g−1 0.4–10 ng g−1 1.6–2.6 ng mL−1

Maragou et al. 2006 Shao et al. 2007a Liu et al. 2008

Milk or soy-based infant formula — USA

5(5) 14(14)

44–113 ng g−1 0.1–13.2 ng mL−1

Kuo and Ding 2004 Biles et al. 1997

10(9)

10–43 ng g−1

Goodson et al. 2002

21(15)

0.7–30.5 ng g−1

Fish USA, Portugal, Canada, South Africa, Thailand, Denmark Japan Spain

4(3)

a

119–129 ng g

Yonekubo and Hayakawa 2008 García-Prieto et al. 2009

Seafood Singapore

5(5)

13–213 ng g−1

Basheer et al. 2004

Meat Australia, New Zealand, USA China

6(2) 20(8)

29–98 ng g−1 0.3–7.1 ng g−1

Thomson et al. 2005 Shao et al. 2007b

Eggs China

10(1)

0.5 ng g−1

Shao et al. 2007a

Soup and sauces Australia, New Zealand United Kingdom Japan

8(4) 10(4) 8(8)

11–21 ng g−1 8–21 ng g−1 0.9–235.4 ng g−1

4(4)

9.6–37.6 ng g−1

Thomson et al. 2005 Goodson et al. 2002 Yonekubo and Hayakawa 2008 Braunrath 2005b

3–33 ng g−1

Inoue et al. 2003

Austria, Thailand Honey Japan

a

No. of Samples (No. of positive quantifiable samples)

107(17)

−1

Pet Foods —

26(26)

13–206 ng g−1

Kang 2002a

Wine Austria (vats)

10(8)

0.2–2.1 ng mL−1

Austria (bottles)

48(37)

0.2–1.6 ng mL−1

Brenn-Struckhofova and Cichna-Mark 2006 Brenn-Struckhofova and Cichna-Mark 2006

Soft drinks Austria

7(6)

0.1–3.4 ng mL−1

Braunrath 2005b

Mineral water Japan (polycarbonate bottles)

5(4)

0.67–8.8 μg L−1

Cao and Corriveau 2008

Concentrations refer only to the solid portion of the container.

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Analysis of Endocrine Disrupting Compounds in Food

water purification systems and the materials involved in sample treatment (SPE columns, glassware, plasticware, etc.). Background signals reported for BPA in water purified with Milli-Q systems were very low (∼20 ng L−1) and only detectable with sensitive analytical methods (e.g., instrumental quantitation limit of 5 pg) (Watabe et al. 2004a, b). No background signals have been detected in the analysis of ultra-high-quality waters (Water Pestanal from Riedel-de Haën or those obtained from an Elgastat and a Millipore Milli-Q system) because methods with significantly higher instrumental quantification limits have been used, that is, 10 ng (CarabiasMartinez et al. 2006). Contamination from this source can be removed by filtering the water through a hydrophobic membrane such as an Empore disk. On the other hand, contamination coming from the SPE step (around 0.04 μg L−1) mainly arises from cartridges (e.g., Oasis HLB from Waters and Bond Elut Certify from Varian) (Inoue et al. 2000) and the glass syringes used for sample loading (Carabias-Martínez et al. 2006). The contamination derived from cartridges has been related not to the material itself, but to the manufacturing process, whereas that found from syringes was identified to arise from the adhesive used to fix the needle. These contamination sources were prevented by prewashing the cartridges (∼15 mL of methanol) and by using a peristaltic pump with Viton tubes from DuPont. A general recommendation to avoid BPA contamination is to use heat-treated glassware (4 h at 400°C) and solvent-washed materials.

Sample preparation There are a wide variety of BPA-containing foods including fresh (vegetables, fruits), canned solid (meat, fish, infant formula), and canned liquid (soft drinks, soups, sauces, mineral water, milk) samples. The solid samples are usually first homogenized and the liquid ones are filtered and/or centrifuged.

Special treatments may be required depending on matrix composition; for example, carbonated beverages are degassed, samples with high protein content may require their removal by precipitation, and fish tissues are crushed and freeze-dried before homogenization. Canned foods containing both liquid and solid portions are usually filtered and treated separately.

Sample extraction Solvent extraction and SPE are the most common techniques for the extraction of BPA from solid and liquid samples, respectively. However, other emerging techniques (e.g., MAE, microwave-assisted extraction; PLE, pressurized liquid extraction; SPME, solidphase microextraction; SBSE, stir bar sorptive extraction; MSPD, matrix solid-phase dispersion) (Figure 15.2), although rarely used so far, are interesting alternatives in terms of sample size, automation, and solvent consumption. A good knowledge of the interactions between extractant and BPA is important for setting up an efficient extraction scheme. The properties of BPA that are of interest for solvent or sorbent selection are its weak acidity (pKa 9.7), moderate hydrophobicity (octanol–water partition coefficient, log KOW 3.3), and the presence of hydrogen acceptor/ donor groups in its structure. In general, extraction methods are matrix dependent, each food type requiring a combination of different techniques. Thus, more suitable sample extraction strategies should be developed to make these methodologies more reliable and widely applicable.

Solvent-based extraction Solvent extraction (SE) is by far the most commonly used technique for the isolation of BPA from solid foodstuffs, whereas liquid– liquid extraction (LLE) has been used to a lesser extent than SPE for liquid foods. Most

Bisphenol A

methods have been proposed for specific food types such as fish (Munguía-López et al. 2005; Gyong et al. 2007), fruits and vegetables (Yoshida et al. 2001; Kang et al. 2006), infant formula powders (Kuo and Ding 2004), or pet foods (Kang and Kondo 2002a), among others. However, a few approaches covering a wide range of matrices have also been reported. In this regard, Goodson et al. proposed a method for the extraction of BPA and isomers of bisphenol F from a variety of canned products, including fish, fruits, vegetables, beverages, soups, desserts, infant formula, meat, and pasta (Goodson et al. 2002), which was subsequently applied by other authors (Thomson and Grounds 2005). As a general rule, repeated solvent extractions are required in solvent-based methods, acetonitrile being preferred for solid foods. Acetone, methanol, and ethanol may also extract BPA efficiently. Regarding liquid foods, ethyl acetate, chloroform, or dichloromethane can be used. Typical overall solvent consumption (including repeated extractions and washing) is in the range of 40–300 mL and extraction times range from 10 to 120 min using stirring or sonication. The addition of sodium anhydrous sulfate (directly to the sample or after extraction) is commonly used to remove residual water in the organic layer. On the other hand, the importance of controlling the enzymatic degradation of BPA during extraction from fresh fruits and vegetables has also been highlighted as cause of poor extraction efficiency (Kang et al. 2006). Degradation was in this case prevented by addition of 0.1 N HCl to the solvent. The application of microwave energy to the sample during extraction (MAE) constitutes a good alternative to the SE of solid and semisolid food samples in terms of solvent consumption and rapidity of extraction. MAE has been applied to the extraction of BPA from fish and seafood (prawns, crabs, cockles, white clams, and squids). Small sample sizes (0.2–1 g of thawed and crushed tissue) were subjected to MAE with 20 mL of

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dichloromethane/methanol (2 : 1 v/v) or 10 mL (20% water content) of tetramethylammonium hydroxide (TMOH) and 1 mL n-nonane for 15–20 min. The cleanup was made with SPE cartridges (Sep-Pak NH2 and Oasis-HLB) followed by evaporation and reconstitution prior to LC-MS (Pedersen and Lindholst 1999) or GC-MS (Basheer et al. 2004). Average recoveries were in the ranges of 47%–49% and 78%–79% for liver and muscle tissue, respectively from rainbow trout and 92%–111% for the GC-MS-based method. PLE or accelerated solvent extraction (ASE) uses solvents at elevated temperatures (40–200°C) and pressures (1000–2500 psi) in order to enhance solvation properties and increase extraction rates. Its application to food analysis combined with LC-MS is increasing. The solvents used in these methods have been acetone-n-hexane (1 : 1, v/v) for fish liver (Tavazzi et al. 2002), dichloromethane for meat products (pork, meat, rabbit, duck, and chicken) (Shao et al. 2007b), and methanol for cereals (Carabias-Martínez et al. 2006). Sodium sulfate and diatomaceous earth have been used as dispersing agents. Recoveries were quantitative for meat and cereals, being in the ranges of 91%–100% and 81%–104% (RSD 4%–9%), respectively, whereas lower recoveries and a higher standard deviation were obtained for fish liver; namely, a value of 53% (RSD 20%) was reported. Recently, the microextraction of BPA from both solid and liquid foods using supramolecular solvents has been reported (García-Prieto et al. 2008, 2009; BallesterosGómez et al. 2009). Supramolecular solvents are water-immiscible liquids made up of large surfactant aggregates dispersed in a continuous phase (usually water). They spontaneously form in micellar or vesicular aqueous or hydro-organic solutions by the action of an external stimulus (e.g., temperature, electrolyte, pH, solvent), which induces the formation of larger aggregates, often keeping the

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morphology, and causes their separation from the bulk solution by a phenomenon named coacervation. Supramolecular solvents have two outstanding properties for microextractions. The first derives from the special structure of the ordered aggregates that constitute the solvants. Thus, they have regions of different polarity that provide a variety of interactions for analytes. The type of interaction may be tuned, varying the hydrophobic or the polar group of the surfactant; in theory one may design the most appropriate extractant for a specific application because amphiphiles are ubiquitous in nature and synthetic chemistry. A second major feature of supramolecular solvents is the high concentration of amphiphiles, and therefore binding sites, they contain (typically 0.1–1 mg μL−1). This characteristic permits the achievement of high extraction efficiencies using low extractant volumes, which is requisite in microextractions. Additional interesting properties for extractions include nonvolatility and nonflammability, which permit the implementation of safer processes, and the use of self-assembly-based synthetic procedures that are within everyone’s reach. Application of supramolecular solvents to BPA microextractions has involved the use of reverse micelles of decanoic acid, which spontaneously coacervate in a water/THF (95 : 5, v/v) mixture. Driving forces for the extraction are Van der Waals interactions between the hydrophobic region of BPA and the surfactant tails at the micellar surface, and hydrogen bonds between the polar head groups and the hydroxyl groups of BPA at the micellar core. BPA was efficiently extracted in 350–550 μL of coacervate from 200 to 400 mg of canned fruits and vegetables (García-Prieto et al. 2008), fatty foods (García-Prieto et al. 2009), and 36–38 mL of beverages (Ballesteros-Gómez et al. 2009). Methods were fast (15 min to reach extraction equilibrium conditions in solid samples) and did not require cleanup of the supramolecular extract, which was directly analyzed in LC-MS/MS for beverages and LC-FL for

solid foods. Recoveries ranged between about 81% and 99% (method precision, RSD: 2%–7%).

Solid-phase extraction (SPE) SPE is the most popular and well-established technique for the extraction of BPAcontaining liquid foods. Recent developments in this field have mainly been related to the use of new sorbent materials (e.g., restricted access materials [RAMs] and molecularly imprinted polymers [MIPs]) and miniaturized techniques (e.g., solid-phase microextraction, SPE; stir bar sorptive extraction, SBSE; and matrix solid-phase dispersion, MSPD), which have the potential to improve extraction efficiencies and extend the scope of SPE. In general, the use of SPE for isolation of BPA from liquid foods offers two advantages over LLE; namely, higher selectivity and lower solvent consumption, whereas concentration factors, enhanced by solvent evaporation, are similar for both techniques. Great care must be taken regarding the small particulates present in the extracts, which can produce low recoveries and irreproducibility by adsorption of analytes or clogging, and usually, dilution and filtration are required. Both nonselective and selective sorbents have shown excellent efficiency for the extraction and cleanup of BPA from foodstuffs. Divinylbenzene/N-vinylpyrrolidone copolymer (OASIS HLB from Waters, 30–200 mg) has been the most commonly used sorbent for extraction of BPA from a wide array of foods, for example, the aqueous portion of canned foods and vegetables (Yoshida et al. 2001), drinking water and soda beverages (Shao et al. 2005), and human colostrum (Kuruto-Niwa et al. 2007). The hydrophilic N-vinylpyrrolidone polymer affords good wettability and acts as a hydrogen acceptor, whereas the hydrophobic divinylbenzene polymer provides reversed-phase retention for BPA. Other sorbents, such as chemically bonded reversed-phase silica (C18) (Maragou

Bisphenol A

et al. 2006), polystyrene-divinylbezene (PSDVB) (Inoue et al. 2003), and multimode phases (Isolute multimode cartridges) (Kang and Kondo 2002b) have also been proposed for the isolation of BPA, recoveries being always higher than 85%. The application of highly selective SPE sorbents, such as RAMs and MIPs to BPA extraction in food analysis, is a promising area but a rather limited one so far. These smart sorbents permit one to carry out both extraction and cleanup in one step. RAMs are used to remove macromolecules from samples, such as lipids and proteins, on the basis of size exclusion, and low molecular mass analytes are concentrated by hydrophobic, ionic, or affinity interactions. A RAM (LiChrosphere RP-18 ADS from Merck) has been proposed for the online SPE-LC-MS/ MS analysis of BPA, other phenolic compounds, and triclocarban in breast milk (Ye et al. 2006). The RAM consists of a bonded reversed-phase covering the internal pore and a surface modified with hydrophilic groups (glycerylpropyl, i.e., diol moieties), which acts as a physical barrier (pore size 6 nm) that excludes the macromolecules. MIPs provide unique selectivity on the basis of molecular recognition. They provide some advantages over immunosorbents, such as stability against organic solvents, strong acids and bases and heating, longer sample volumes, reusability, and no dependence on antibody production. To the best of our knowledge only one such application has been developed for extraction of BPA from food (Martin-Esteban and Luis-Tadeo 2006). The MIP was synthesized from methacrylic acid as functional monomer, trimethylolpropane trimethacrylate (TRIM) as cross-linker, 2,2′azobis methylbutyronitrile (AIMN) as initiator, BPA as template, and toluene as solvent. BPA was determined in the filtered aqueous phase of canned foods (recovery 78%; RSD 10%, n = 3) free of coextractives. The main drawback of MIPs continues to be their production, which is too time consuming, complex, and expensive for routine analysis.

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Sorptive microextraction techniques, such as SPME and SBSE, reduce solvent consumption and sample handling and offer the possibility of automation; however, their application to the extraction of BPA from food is still limited. The SPME device consists of a fused-silica fiber coated with an appropriate stationary phase attached to a modified microsyringe, whereas SBSE uses a stir bar in a sealed glass tube that is coated with polydimethylsiloxane (PDMS). The fiber or the bar can be suspended in the vapor phase above the liquid sample (headspace [HS]) or directly immersed into the sample (direct immersion [DI]). Then, desorption is carried out thermally, when coupling with GC, or with a solvent, when coupling with LC. DI-SPME followed by GC-MS has been applied mostly to determine BPA in simple matrices, such as aqueous food simulants (Salafranca et al. 1999), water from plastic containers, and tableware (Chang et al. 2005). DI-SPME has also been proposed prior to LC-FL for the screening of BPA in the liquid portion of canned foods (Nerín et al. 2002). A number of sorbent materials with the capability to withstand high injector temperatures (polydimethylsiloxane, PDMS; carboxen/ PDMS; PDMS/divinyl benzene, DVB; carbowax CW/DVB; and polyacrylate, PA) were tested for the extraction of BPA by DISPME-GC. Polyacrylate fibers gave better results because they have a polarity similar to BPA. Among the coating materials tested for extraction of BPA by DI-SPME-LC, polar fibers made of Carbowax gave the highest recoveries. Maximum sorption occurred at neutral pH, and salt was added at percentages of 7.5%–15.4 w/w to enhance extraction efficiency. Most of these methods, although sensitive enough for screening—quantitation limits were 3.8 mg L−1 (Nerín et al. 2002) and 0.01–0.02 mg L−1 (Salafranca et al. 1999; Chang et al. 2005) also presented serious drawbacks. Thus, the automation of SPME with LC-FL through the interface supplied by Supelco was not successful in terms of sensitivity and peak resolution. In these

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Analysis of Endocrine Disrupting Compounds in Food

terms, in-tube SPME, which uses an innerwall coated capillary, is most suitable for automation and has been successfully applied to the extraction of BPA and other estrogens from environmental waters (Wen et al. 2006) but not in food samples as yet. Additional weak points for both SPME-LC-FL and SPME-GC-MS methods were the reproducibility (13%–22%) and the low recoveries (7%–65%) resulting from the competition between BPA and matrix components for binding to the limited volume of stationary phase, which made necessary the use of the standard addition method for quantitation. In fact, the use of DI-SPME for more complex food matrices requires exhaustive cleanup to ensure the reproducibility and the robustness of the fibers, which can be affected by adsorption of proteins or clogged by particles. In these terms, a convenient approach consisting of a DI-SPME-LC-FL method has been recently proposed for the analysis of BPA milk (Liu et al. 2008). The extraction from this complex matrix requires a high dilution factor (20-fold with water) and the acidification and addition of methanol for protein precipitation. Satisfactory recoveries were obtained for pure milk (93%–101%) and soybean milk (94%–102%). The quantitation limit of the method was 0.5 ng mL−1. Regarding SBSE, because PDMS is the only commercially available coating, its application to semipolar compounds such as BPA requires derivatization, namely with acetic anhydride. Despite the fact that new SBSE coatings are necessary to extend the scope of this technique, few coatings have been reported up to now. In this respect, a novel SBSE coating based on polydimethylsiloxane/β-cyclodextrin (PDMS/β-CD) has been recently applied to the extraction of BPA (and other estrogenic compounds) from drinking water and leachate from disposable dishware before LC-FL (Hu et al. 2007). The presence of the cyclodextrin is permitted to enhance the recoveries of polar compounds due to the high number of hydroxyl groups

of β-CD and the selectivity given by its molecular recognition ability. The LOD for BPA was 8 ng L−1, with recoveries between 86% and 116% (RSDs: 0.7%–10.7%). Other SPE-convenient formats, such as matrix solid-phase dispersion (MSPD), have also been proposed for analysis of BPA in food. MSPD overcomes the disadvantages of the slow extractions and low breakthrough volumes of conventional SPE cartridges and simplifies the extraction of solid samples. An example is the extraction of BPA, nonylphenol, and octylphenol from eggs and milk (Shao et al. 2007a) before LC-MS/MS. The samples (1 g) were blended with 1 g of C18 powder (5 min), packed, and eluted with 10 mL of ethanol. This sorbent gave better results than graphite carbon black because of the strong adsorption of BPA to this material. A second SPE with aminopropyl cartridges was necessary for lipid removal. Mean recoveries for BPA ranged from 82% to 91% (RSD: 4%–6%).

Sample cleanup The extracts containing BPA are commonly subject to extensive cleanup, and in this respect liquid–liquid extraction and SPE are preferred. Lipid removal is essential for fatty foods (e.g., fish, meat) because they can significantly reduce the analytical performance of LC and GC. Lipidic material affects the active surface of the stationary phase in LC and degrades the resolution power of the column. In GC-MS, lipids accumulate in the injection port, column, and ion source. Fat removal is mainly performed by extraction with n-heptane (Goodson et al. 2002), trimethylpentane (Thomson and Grounds 2005), or n-hexane (Munguía-López et al. 2005), or by freezing the lipids in the extract at −24°C for 40 min followed by filtration (Gyong et al. 2007). On the other hand, protein precipitation in dairy products, such as human colostrum (Kuruto-Niwa et al. 2007), is carried out with acetonitrile or 2-propanol.

Bisphenol A

The use of SPE for cleaning the extracts, and the subsequent evaporation, reconstitution, and filtration of the eluates are steps required in most of the sample treatment procedures. Oasis HLB is by far the preferred sorbent for sample cleanup. Florisil 60, aminopropyl, and immunoaffinity columns (IAC) have also been applied in this context. OASIS HLB efficiently removes hydrophilic and lipophilic interferences and it has been applied to the cleanup of a variety of foods after solvent extraction, including fish (Gyong et al. 2007), fruits and vegetables (Yoshida et al. 2001), and canned foods (Kang et al. 2006). A second cleanup step with a normal-phase SPE sorbent (Florisil 60, magnesium silicate) was necessary in some applications (Yoshida et al. 2001; Gyong et al. 2007). Because retention of BPA on Florisil mainly occurs through adsorption, its application requires previous evaporation and reconstitution of the extracts in a nonpolar organic solvent such as n-hexane. Although overall recoveries using these sample treatments are normally quantitative (well above 75%), low recoveries due to matrix–analyte interactions (e.g., <50% in Thomson [2005]) or the presence of hydrolyzed enzymes influencing the SPE (e.g., <18% with RSD of 24% [Kuo and Ding (2004)] have been reported. Sol-gel immunoaffinity columns have also been used for the cleanup of liquid foods or crude extracts prior to LC-fluorescence detection of BPA in a variety of fatty foods (e.g., tuna, cream), fruits and vegetables, soft drinks, and wine (Braunrath and Cichna 2005a, b; Brenn-Struckhofova and CichnaMarkl 2006). Substances showing high crossreactivity (>1%) were efficiently separated by the LC system and did not affect BPA determination. However, recoveries strongly depended on food composition (e.g., 53% in peas, 75% in peaches, 27% in goulash, 103% in a lemon soft drink, and 74%–81% in wines), and RSD values between 1% and 21% were obtained. The variable and low recoveries obtained for IAC were due to the

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matrix-dependent nature of the previous solvent-based BPA extraction and the influence of coextracted matrix components on the BPA–antibody interactions. In general, food samples have to be treated to some extent in order to make them compatible with IACs. Detailed information about sample treatment for BPA analysis is summarized in Table 15.2.

Separation and detection Due to the complexity of food matrices and the low levels at which BPA is present, its determination requires methods highly sensitive and selective. Although the SML set by the EU Commission is relatively high (600 ng g−1), the reported effects of BPA at trace levels have led to the development of analytical methods with LODs low enough to assess the risk of human exposure to BPA in low doses. At present, LC-fluorescence detection (LC-FL) is still frequently used and it gives satisfactory quantitative results, but more complex techniques, that is, LC-MS and GC-MS, are becoming more attractive because they provide more reliable results. Good sample preparation is still necessary when using MS detection, because matrix components can affect ionization efficiency and background noise, and consequently sensitivity. Furthermore, clean extracts are preferred in general for good instrument maintenance and to extend the column life. LC-MS offers the advantage of simplicity over GC-MS for which a derivatization step is necessary, thus making methods labor intensive and introducing new sources of errors, mainly due to contamination. However, GC is a good alternative because it provides higher peak resolution and better sensitivity. In this regard, Stuart et al. (2005) compared the instrumental LODs obtained by different techniques for BPA; the values were 0.004 ng for GC-MS using methyl derivate, 1.6 ng for LC-UV (275 nm), and 1.0 ng for LC-ESI(-)-MS.

358 Freeze-drying (for tuna fish) SE with acetonitrile (20 mL) for food simulant or methanol (100 mL) for tuna fish Cleanup with hexane

LC-FL (275/300 nm) (confirmation by GC-MS)

GC-MS

Solvent extraction with methanol (70 mL) under sonication (30 min) Delipidation (freezing lipid) SPE cleanup (Oasis HLB and Florisil cartridges) Derivatization (sylilation with MTBSTFA)

Fish (10 g)

Fatty-food simulant (commercial sunflower oil) (5 g) Tuna fish (7 g)

LC-FL (276/306 nm)

LC-FL (275/300 nm)

LC-UV (228 nm, DAD)

GC-MS

GC-MS

Separation and Detection

Supramolecular solvent extraction

Solid portion: SE with acetonitrile (130 mL) followed by cleanup with hexane and subsequent SPE (Florisil) Liquid portion: dilution 1 : 1 with water, SPE extraction and subsequent SPE cleanup (Florisil) SE with acetone (70 mL) under mechanical shaking (10 min). SPE cleanup (Oasis HLB and Florisil)

SE (except for beverages) with 40 mL of acetonitrile (except for infant formula, 20 mL) Cleanup of fatty matrices with heptane Derivatization (acetic anhydride) The same steps as those proposed by Goodson et al. (2002)

Sample Treatment

Fruits and vegetables (0.3 g) 4-tert-Butylphenol 4-n-Butylphenol 4-Pentylphenol 4-n-Hexylphenol 4-tert-Octylphenol 4-n-Heptylphenol Nonylphenol 4-Octylphenol IS: BPA-d16

IS: BPA-d14

Vegetables, fruits, fish, soup, sauces, canned meat, spaghetti, baked beans, infant foods (20 g) Beverages (50 mL) Fruits, vegetables and corn (5 g solid portion, 10 mL liquid portion)

Fruits and vegetables (5 g)

Bisphenol F IS: BPA-d14

Analytes Other than BPA

Vegetables, desserts, fish, soup (20 g) Infant foods (10 g) Beverages (50 mL)

Sample Type (size)

Table 15.2. Procedures and figures of merit for the determination of BPA in foods.

LR: 5–1200 ng mL−1 LOQ: 10 ng g−1 (food simulant) 7.1 ng g−1 (tuna fish) R (mean): 91(food simulant), 77 (tuna fish) RSD (mean): 5(food simulant), 9 (tuna fish)

LR: 0.05–1 μg mL−1 LOQ: 10 ng g−1 (solid portion), 5 ng mL−1 (aqueous portion). R (mean in corn infusion, n = 3): 87–90 RSD (mean in corn infusion, n = 3): 3–4 LR: 10–1000 ng mL−1 LOD: 1.3 ng g−1 R (n = 5): 82–100 RSD (n = 5): 4–22 LR: 0.14–20 ng LOD: 1.3 ng g−1 R (n = 3): 81–96 RSD (n = 6): 2.8 LR: 0.5–50 ng g−1 LOD: 0.41 ng g−1 R (n = 7): 105–120 RSD (n = 7) 3–14

Goodson et al. 2002

LR: 5–700 ng g−1 LOD: 2 ng g−1 R: 81–103 RSD: 4.6 (mean in a can of salmon, n = 6) LOQ: 10 ng g−1 (samples <1%fat), 20 ng g−1 (samples >1% fat) R: 42–112

Munguia-López et al. 2005

Gyong et al. 2007

García-Prieto et al. 2008

Kang et al. 2006

Yoshida et al. 2001

Thomson and Grounds 2005

Reference

Instrumental Linear Range (LR), Method LOD or LOQ, Recoveries (R, %) and aRSD (%) for BPA

359

4-tert-Octylphenol IS: 13C12-BPA 4-tert-Octylphenol Nonylphenol IS: 4-n-nonylphenol

Breast milk (100 μL)

Drinking water (2 L) and soda beverages (50 mL)

4-n-nonylphenol 4-tert-butylphenol 2,4-dichlorophenol 2,4,5-trichlorophenol Pentachlorophenol 4-tert-butylbenzoic acid Octylphenol Nonylphenol IS: 4-n-Nonylphenol

Cereals (2.5 g)

Homogenization PLE (dichloromethane, 3 cycles, 3 min, 100°C, 1500 psi) SPE cleanup (amino-propyl cartridge)

Sample grinding PLE (methanol, one cycle, 10 min, 120°C and 1600 psi). SPE cleanup (Oasis HLB)

Degassing Dilution with PBS Filtering and pH set at 7 Sol-gel precolumn-IAC for simultaneous extraction and cleanup Dilution with water (50 mL) -SPE (PS-DVB) extraction

SE with ethanol-water (50 : 50, v/v) (10 mL) under stirring with a magnetic bar (10 min) followed by ultrasonication (2 h, 4°C). Ultracentrifugation Cleanup of crude extract by SPE (C18) Derivatization (sylilation with BSTFA : TMCS : DTE) Enzymatic hydrolysis Protein precipitation Online SPE extraction (RAM, LiChrosphere RP-18 ADS) SPE extraction (Oasis HLB)

Sample Treatment

LC-(ESI-)MS/ MS

LC-(ESI-)MS

LC-FL (275/300 nm)

LC-FL (275/305 nm)

LC-(ESI-)MS/ MS

LC-(APCI-)MS

GC-MS

Separation and Detection

LR: 1–500 ng mL−1 LOD: 0.3 ng g−1 R (n = 5) : 92–97 RSD (n = 5): 2.7–14.7

LR: 25–500 ng mL−1 LOD: 2 ng g−1; R (n = 6):99.9–103 RSD (n = 6): 5.3–6.6 LR: 0.08–0.8 μg g−1 (standard addition) LOD: 43 ng g−1 R (n = 6): 81–104 (PLE step) RSD (n = 6): 5.3–6.6 (PLE step)

Ye et al. 2006

LR: 0.1–100 ng mL−1 LOD: 0.28 ng mL−1 R (n = 5): 97–106 RSD (n = 50): 8.2–11.4 LR: 1 to 500 ng mL−1 LOD: 0.01 ng mL−1 (mineral drinking water),0.6 ng mL−1 (soda beverages) R (n = 5): 82.1–96.5 RSD (n = 5): 2.9–7.1 LR: 0.5–100 ng mL−1 LOD: 0.1 ng mL−1 R (n = 3): 74–81 RSD (n = 3)=8–11

Shao 2007b

Carabias-Martinez et al. 2006

Inoue et al. 2003

BrennStruckhofava 2006

Shao et al. 2005

Kuo and Ding 2004

Reference

LR: 0.01–50 μg mL−1 LOD: 1 ng g−1 R (n = 3): 18–97 RSD (n = 3): 5–24

Instrumental Linear Range (LR), Method LOD or LOQ, Recoveries (R, %) and aRSD (%) for BPA

a Spiked samples, RSD as repeatability. Abbreviations: IS, internal standard; SE, solvent extraction; SPE, solid-phase extraction; UV, ultraviolet detection; DAD, diode array detection; FL, fluorescence detection; MTBSTFA, N′,N′-methyl-(tert-butyldimethylsilyl) trifluoroacetamide; BSTFA, N-O-bis(trimethylsilyl) trifluoroacetamide; TMCS, trimethylchlorosilane; DTE, 1,4-dithioerythritol; RAM, restricted access material; APCI, atmospheric pressure chemical ionization; ESI, electrospray ionization; PBS, phosphate-buffered saline; IAC, immunoaffinity chromatography; PS-DVB, polystyrene divinilbenzene; PLE, pressurized liquid extraction.

Meat (10 g)

Bisphenol F

Honey (10 g)

Wine (25 mL)

Daidzein Genistein IS: Chrysene-d12

Analytes Other than BPA

Powdered infant formula (0.5 g)

Sample Type (size)

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Analysis of Endocrine Disrupting Compounds in Food

The use of an internal standard (IS) is common in MS to correct matrix effects leading to suppression or enhancement of the signal or losses during sample preparation, which results in low recoveries. Deuterated BPA-d16 and BPA-d14 are the most used surrogates in GC-MS, whereas for LC-MS, 4-nonylphenol (when alkylphenols were also determined) and isotope-labeled 13C12-BPA have been also employed. The importance of using an IS was highlighted by Maragou et al. (2006), who could overcome the losses caused by signal suppression (∼20%) and those occurring during sample preparation (∼28%) by using BPA-d16. In this way, the mean relative recovery of the method was 101% (mean absolute recovery of 52%). ELISA, although rarely used so far, is a convenient approach for screening of BPA in food, offering a cheaper and faster alternative to common methods for this purpose. Other detection techniques, such as ultraviolet detection (diode array, quantification at 228 nm) (Yoshida et al. 2001) and electrochemical detection have been reported to a lesser extent. Detailed information about the figures of merit for some of the analytical methods used for determination of BPA in food is summarized in Table 15.2.

Liquid chromatography (LC) LC of BPA is mainly carried out in reversedphase C18 columns, water and acetonitrile being the most common binary solvents when fluorescence detection is used and water and methanol the preferred ones for MS detection. Elution conditions highly depend on the analytes to be determined as well as the BPA and the complexity of the matrix, with run times ranging between 15 and 40 min. Fluorescence detection BPA shows native fluorescence with excitation and emission wavelengths at 275 and

305 nm, respectively. Typical instrumental quantitation limits for BPA by LC-fluorimetry are between 5 and 50 ng mL−1. The identification of BPA in the sample is based only on retention times, so the possibility of false positives caused by other fluorescent food migrants from can coatings, for example, bisphenol A diglycidyl ether (BADGE), bisphenol F diglycidyl ether (BFDGE), or novolacs glycidyl ethers (NOGE), should be always considered. Sometimes, the subsequent injection of the extracts in LC-MS (Inoue et al. 2003) or in GC-MS (MunguíaLópez et al. 2002) has been carried out for confirmation. A variety of LC-FL methods giving consistent results have been proposed in literature for determination of BPA in foods, for example vegetables, fruits, wines, fish and honey (see Table 15.2), providing detection limits in the ranges 0.1–2 ng mL−1 and 1–5 ng g−1.

Electrochemical detection Electrochemical detection (ED) of BPA is based on the electroactivity of the phenolic groups present in the molecule. Instrumental detection limits obtained for BPA with LC-ED are 3000 and 200 times lower than those obtained with UV and FL detectors, respectively (Inoue et al. 2000). The pH and electrolyte content of the mobile phase influence the electron-transfer rate constants and have to be optimized to achieve maximal sensitivity. To prevent large equilibrium times, isocratic elution is recommended, which is a major drawback of ED. LC-ED has been used for the analysis of BPA in food simulants (water, acidified water, and water-ethanol) using a homemade electrochemical detector (D’Antuono et al. 2001). The quantification limit of the method was 4 pg after concentration of the sample. Despite its proven suitability for these samples, LC-ED has not yet been applied to common complex food matrices.

Bisphenol A

Mass spectrometric detection The LC-MS analysis of BPA is carried out using atmospheric pressure ionization interfaces, namely electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), both in negative mode. ESI is more frequently used than APCI because it generally provides better sensitivity. Instrumental quantitation limits for BPA of 50 pg (Maragou et al. 2006) and 207 pg (Pedersen and Lindholst 1999) have been reported using ESI(-) and APCI(-), respectively, with a quadrupolar mass analyzer. On the other hand, using ESI(-) and a triple quadrupole mass analyzer the instrumental LOQ can be decreased up to 10 pg (Shao et al. 2005, 2007a; Ye et al. 2006). Ion-trap instruments have been used to a lesser extent, providing higher instrumental quantitation limits (500 pg) (Ballesteros et al. 2009). The response in ESI-MS for BPA is strongly dependent on the mobile phase composition. Methanol and water are preferred over mixtures of acetonitrile-water, the former giving higher response for BPA because of the lower boiling point, which favors the desolvation of electrospray droplets. The response in acetonitrile-water can be increased by three- and fourfold with the addition of modifiers, namely 0.5% (Benijts et al. 2004) and 0.01% ammonia or 0.01% acetic acid (Maragou et al. 2006). Contrary to these studies, the presence of additives in methanol-water mobile phases was found to decrease the response for BPA. Independently of the type of analyzer and ionization source, the most abundant ion in the BPA mass spectrum, and therefore that used for quantitation, is [M-H]− m/z 227. The mass spectrum obtained with quadrupole instruments also contains the fragment ions m/z 211 and 212, formed by the additional loss of oxygen, [M-H-O]−, and a methyl radical, [M-H-CH3]−•, respectively. Using mobile phases of acetonitrile–0.01% NH3 in water, an ion with m/z 113 has also been

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detected and related to the loss of both acidic protons [M-2H]2−. On the other hand, in MS/ MS detection, the [M-H-CH3]−• m/z 212 is the most abundant product ion and thus the one used for confirmation and/or quantification of BPA. Other fragments of lower relative abundance have been reported in the MS2 ion-trap spectrum, namely the ion [M-H-C6H5OH]− m/z 133, coming from the cleavage of the hydroxybenzyl group, and the ion [M-H-C9H10O]− m/z 93, formed by the loss of hydroxyphenyl propyl. LC–ESI(-)-MS or MS/MS methods have been developed for the determination of BPA in cereals, water, beverages, meat, milk, and eggs, with method detection limits in the ranges 0.7–43 ng g−1 and 0.001–0.3 ng g−1 for MS and MS/MS detection, respectively. On the other hand, two methods using APCI(-) have been proposed for the determination of BPA in fish and breast milk. LODs were in the range 0.1–50 ng g−1. More detailed information is given in Table 15.2.

GC-MS GC-MS with electron impact (EI) ionization has been widely used for the confirmation of BPA in food analysis (Biles et al. 1997; D’Antuono et al. 2001; Munguía-López et al. 2005). In this case, derivatization of BPA is not required. The base peak in the spectrum corresponds to the loss of a methyl group ([C14H13O2]+, m/z 213) from the molecular ion ([C15H16O2]+, m/z 228). This can be explained on the basis of resonance stabilization of the resulting tert-benzylic carbocation. Another minor fragmentation pathway takes place through the loss of one of the aryl groups to give a tert-benzylic carbocation ([C9H8O]+, m/z 135) with the subsequent loss of methane to give a fragment ion at m/z 119. However, the derivatization of BPA becomes inevitable to achieve good GC separation and sensitive (sub-ng g−1) quantitation. Silylation and acetylation have been by far the most frequently used derivatization

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Analysis of Endocrine Disrupting Compounds in Food

procedures. Silylation of the active hydrogens of BPA is mainly made using N-Obis(trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% of trimethylchlorosilane (TMCS) (Basheer et al. 2004; Kuo and Ding 2004) to favor the formation of a single derivative. N′,N′-methyl-(trimethylsilyl) trifluoroacetamide (MSTFA) and N′,N′-methyl(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) (Gyong et al. 2007) have also been employed for silylation, the latter giving a derivative with higher storage stability. Acetylation of the hydroxyl groups of BPA with acetic anhydride (Goodson et al. 2002) or trifluoroacetic anhydride (Varelis and Balafas 2000) is another frequent procedure used to obtain BPA derivatives for GC-MS. The derivatization of the IS (e.g., BPA-d14) is essential to maintain its isotopic purity during chromatographic separation on a fused-silica capillary column (Varelis and Balafas 2000), since it degrades due to the deuteron–proton (2H—H) exchange in the aromatic portion of the molecule. In this respect, the use of O-bis(trifluoroacetyl) derivative is preferred to prevent 2H—H exchange in the column, because its stability is higher, and in addition, its higher molecular mass and the monoisotopic nature of fluorine make this derivative more sensitive. GC-MS methods have also been applied to a wide array of foods (e.g., fish, vegetables, infant formula, etc.). Although GC-MS instrumental limits are lower than those obtained by LC-MS, method detection limits were similar (0.4–2 ng g−1 and 0.4–6.3 ng L−1, respectively), probably due to the fact that concentration factors are limited by the higher degree of cleanup needed for GC compared with LC.

Immunochemical methods Immunoassays are easy to perform, they require neither qualified personnel nor expensive equipment, and they provide good sensitivity and specificity, characteristics that made them suitable for the development of rapid

methods for routine analysis. BPA, due to its small size, is not able to cause an immune response itself and needs to be conjugated with a protein to form a complete antigen. With this aim, the derivatized BPA or a structural analog (e.g., 4,4-bis[4-hydroxyphenyl] valeric acid, BHPVA) is attached to the protein bovine serum albumin (BSA) for the production of antibodies. The application of immunochemical methods to the analysis of BPA in foods is rather recent, and few approaches have been developed so far, all of them applied to liquid foods (mainly milk, water, and food stimulants), using polyclonal mammalian and chicken antibodies in enzyme-linked immunosorbent assays (ELISAs) (Ohkuma et al. 2002; De Meulenaer et al. 2002). The detection limits varied from 0.05 ng mL−1 to 500 ng mL−1, mainly depending on the immunogen and the type of antibody produced. An ELISA commercial kit for BPA (EcoAssay BPA kit supplied by Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan) was used for analysis of human colostrum (Kuruto-Niwa et al. 2007). This kit was able to detect glucuronide-conjugated BPA and free BPA. The mean recovery was 102% ± 19.0%, and the detection limit of the method was 0.3 ng mL−1.

Conclusions Accurate and robust methods that meet the criteria to be used in both the enforcement of legislation and assessment of the risk of human exposure to BPA low doses are already available. However, sample treatment still constitutes the bottleneck of these methods, rendering them long, labor intensive, and the main cause of analytical errors. Solvent extraction and SPE are by far the most commonly used extraction techniques for both isolation of BPA and cleanup of matrix components. Many other alternatives, which offer advantages in terms of rapidity, simplicity, and solvent consumption (e.g., SPME, SBSE, etc.), are being developed, but they still present serious drawbacks that restrict their

Bisphenol A

application. The use of alternative extractants, such as supramolecular solvents, is another promising area under research, which has already proved to efficiently extract BPA from a variety of matrices. Fluorometric and mass spectrometric detection combined with LC are predominant in the analysis of BPA in food, and undoubtedly, they will continue to play a key role in the future. However, more convenient ELISA methods will probably be developed to cover the current gap in screening methods for BPA in food.

Acknowledgments The authors gratefully acknowledge financial support from Spanish MICINN (Project CT2008-01068). A. Ballesteros-Gómez acknowledges the Spanish MEC for the doctoral fellowship awarded (AP2005–4275).

References Akingbemi, Benson T.; Sottas, Chantal M.; Koulova, Anna I.; Klinefelter, Gary R.; Hardy, Matthew P. 2004. Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells. Endocrinology. 145(2):592–603. Ballesteros-Gómez, Ana; Rubio, Soledad; Pérez-Bendito, Dolores. 2009. Potential of supramolecular solvents for the extraction of contaminants in liquid foods. Journal of Chromatography A. 1216(3):530–539. Basheer, Chanbasha; Lee, Hian K.; Tan, Koh S. 2004. Endocrine disrupting alkylphenols and bisphenol-A in coastal waters and supermarket seafood from Singapore. Marine Pollution Bulletin. 48(11–12):1161– 1167. Benijts, Tom; Lambert, Willy; De Leenheer, Andre. 2004. Analysis of multiple endocrine disruptors in environmental waters via wide-Spectrum solid-phase extraction and dual-polarity ionization LC-Ion TrapMS/MS. Analytical Chemistry. 76(3):704–711. Biles, J.E.; McNeal, T.P.; Begley, T.H. 1997. Determination of bisphenol A migrating from epoxy can coatings to infant formula liquid concentrates. Journal of Agricultural and Food Chemistry. 45(12): 4697–4700. Braunrath, R.; Cichna, M. 2005a. Sample preparation including sol–gel immunoaffinity chromatography for determination of bisphenol A in canned beverages, fruits and vegetables. Journal of Chromatography A. 1062(2):189–198.

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Braunrath, R.; Polidpna, D.; Padlesak, S.; Cichna-Markl, M. 2005b. Determination of bisphenol A in canned foods by immunoaffinity chromatography, HPLC, and fluorescence detection. Journal of Agricultural and Food Chemistry. 53(23):8911–8917. Brenn-Struckhofova, Z.; Cichna-Markl, M. 2006. Determination of bisphenol A in wine by sol-gel immunoaffinity chromatography, HPLC and fluorescence detection. Food Additives and Contaminants. 23(11):1227–1235. Burridge, E. 2003. Bisphenol A: Product profile. European Chemical News. 14–20:17. Cao, Xu-Liang; Corriveau, Jeannette. 2008. Survey of bisphenol A in bottled water products in Canada. Food Additives and Contaminants. 1(2):161–164. Carabias-Martinez, R.; Rodriguez-Gonzalo, E.; RevillaRuiz, P. 2006. Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography–mass spectrometry: Study of background contamination. Journal of Chromatography A. 1137(2):207–215. Chang, Chia M.; Chou, Chi C.; Lee, Maw R. 2005. Determining leaching of bisphenol A from plastic containers by solid-phase microextraction and gas chromatography–mass spectrometry. Analytica Chimica Acta. 539(1–2):41–47. Chen, Min-Yu; Ike, Michihiko; Fujita, Masanori. 2002. Acute toxicity, mutagenicity, and estrogenicity of bisphenol A and other bisphenols. Environmental Toxicology. 17(1):80–86. D’Antuono, Alejandra; Campo Dall’Orto, Viviana; Lo Balbo, Alfredo; Sobral, Santiago; Rezzano, Irene. 2001. Determination of bisphenol A in food-simulating liquids using LCED with a chemically modified electrode. Journal of Agricultural and Food Chemistry. 49(3):1098–1101. De Meulenaer, Bruno; Baert, Katleen; Lanckriet, Heikki; Van Hoed, Vera; Huyghebaert, Andre. 2002. Development of an enzyme-linked immunosorbent assay for bisphenol A using chicken immunoglobulins. Journal of Agricultural and Food Chemistry. 50(19):5273–5282. European Commission (EC). 2004. Directive 2004/19/ EC, of 1 March 2004, relating to plastic materials and articles intended to come into contact with foodstuffs. Journal of the European Communities. L71(8). European Food Safety Authority (EFSA). 2006. Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (ACF). Opinion on request from the Commission related to 2,2-bis(4-hydroxyphenyl) propane (Bisphenol A). Question number EFSA-Q2005–100, adopted on 29 November 2006. The EFSA Journal. 428(1). García-Prieto, Amalia; Lunar, Loreto; Rubio, Soledad; Pérez-Bendito, Dolores. 2008. Decanoic acid reverse micelle-based coacervates for the microextraction of bisphenol A from canned vegetables and fruits. Analytica Chimica Acta. 617(1–2):51–58. García-Prieto, Amalia; Lunar-Reyes, María L.; RubioBravo, S.; Pérez-Bendito, María D. 2009. Determination of bisphenol A in canned fatty foods by coacervative microextraction, liquid chromatography

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and fluorimetry. Food Additives and Contaminants. 26(2):265–274. Goodson, A.; Summerfield, W.; Cooper, I. 2002. Survey of bisphenol A and bisphenol F in canned foods. Food Additives and Contaminants. 19(8):796–802. Gyong, Yun G.; Shin, Jeoung H.; Kim, Hye Y.; Khim, Jeehyeong; Lee, Mi K.; Hong, Jongki. 2007. Application of solid-phase extraction coupled with freezing-lipid filtration clean-up for the determination of endocrine-disrupting phenols in fish. Analytica Chimica Acta. 603(1):67–75. Hu, Yuling; Zheng, Yanjie; Zhu, Fei; Gongke, Li. 2007. Sol–gel coated polydimethylsiloxane/β-cyclodextrin as novel stationary phase for stir bar sorptive extraction and its application to analysis of estrogens and bisphenol A. Journal of Chromatography A. 1148(1):16–22. Inoue, Koichi; Kato, Kayoko; Yoshimura, Yoshihiro; Makino, Tsunehisa; Nakazawa, Hiroyuki. 2000. Determination of bisphenol A in human serum by high-performance liquid chromatography with multi-electrode electrochemical detection. Journal of Chromatography B. 749(1):17–23. Inoue, Koichi; Murayama, Shiho; Takeba, Kazue; Yoshimura, Yoshihiro; Nakazawa, Hiroyuki. 2003. Contamination of xenoestrogens bisphenol A and F in honey: Safety assessment and analytical method of these compounds in honey. Journal of Food Composition and Analysis. 16(4):497–506. Integrated Risk Information System (IRIS). 1988. Bisphenol A (CASRN 80-05-7). Washington, DC, U.S. Environmental Protection Agency. Kang, Jeong H.; Kondo, F. 2002a. Determination of bisphenol A in canned pet foods. Research in Veterinary Science. 73(2):177–182. Kang, Jeong H.; Kondo, Fusao. 2002b. Bisphenol A migration from cans containing coffee and caffeine. Food Additives and Contaminants. 19(9):886–890. Kang, Jeong. H; Kondo, F.; Katayama, Y. 2006. Importance of control of enzymatic degradation for determination of bisphenol A from fruits and vegetables. Analytica Chimica Acta. 555(1):114–117. Krishnan, Runa V.; Stathis, Peter; Permuth, Suzanne F.; Tokes, Laszlo; Feldman, David. 1993. Bisphenol-A: An estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology. 132(6):2279–2286. Kuo, Han W.; Ding, Wang H. 2004. Trace determination of bisphenol A and phytoestrogens in infant formula powders by gas chromatography–mass spectrometry. Journal of Chromatography A. 1027(1–2):67–74. Kuruto-Niwa, Ryoko; Tateoka, Yumiko; Usuki, Yasuteru; Nozawa, Ryushi. 2007. Measurement of bisphenol A concentrations in human colostrums. Chemosphere. 66(6):1160–1164. Liu, Xiaoyan; Ji, Yongsheng; Zhang, Haixia; Liu, Mancang. 2008. Elimination of matrix effects in the determination of bisphenol A in milk by solid-phase microextraction-high-performance liquid chromatography. Food Additives and Contaminants. 25(6): 772–778.

Lyons, Gwynne. 2000. Bisphenol A: A known endocrine disruptor, a WWWF European Toxics Programme Report. Survey: WWWF-UK. Maragou, Niki C.; Lampi, Eugenia N.; Thomaidis, Nikolaos S.; Koupparis, Michael A. 2006. Determination of bisphenol A in milk by solid phase extraction and liquid chromatography–mass spectrometry. Journal of Chromatography A. 1129(2):165– 173. Martin-Esteban, Antonio; Luis-Tadeo, José. 2006. Selective molecularly imprinted polymer obtained from a combinatorial library for the extraction of bisphenol A. Combinatorial Chemistry and High Throughput Screening. 9(10):747–751. Morgan, M. 2006. Phenol/acetone-facts, figures and future. Paper presented at 4th ICIS World Phenol/ Acetone Conference, 6–7 June, at Prague, Czech Republic. Munguía-López, E.M.; Gerardo-Lugo, S.; Peralta, E.; Bolumen, S.; Soto-Valdez, H. 2005. Migration of bisphenol A (BPA) from can coatings into a fatty-food simulant and tuna fish. Food Additives and Contaminants. 22(9):892–898. Munguía-López, Elvia M.; Peralta, Elisabeth; Gonzalez-León, Alberto; Vargas-Requena, Claudia; Soto-Valdez, Herlinda. 2002. Migration of bisphenol A (BPA) from epoxy can coatings to jalapeno peppers and an acid food simulant. Journal of Agricultural and Food Chemistry. 50(25):7299–7302. Nerín, C.; Philo, M.R.; Salafranca, J.; Castle, L. 2002. Determination of bisphenol-type contaminants from food packaging materials in aqueous foods by solidphase microextraction–high-performance liquid chromatography. Journal of Chromatography A. 963(1–2): 375–380. Ohkuma, H.; Abe, M.; Ito, A.; Kokado, A.; Kambegawa, A.; Maeda, M. 2002. Development of a highly sensitive enzyme-linked immunosorbent assay for bisphenol A in serum. Analyst. 127(1)93–97. Pedersen, Soren N.; Lindholst, Christian. 1999. Quantification of the xenoestrogens 4-tertoctylphenol and bisphenol A in water and in fish tissue based on microwave assisted extraction, solid-phase extraction and liquid chromatography–mass spectrometry. Journal of Chromatography A. 864(1): 17–24. Salafranca, Jesús; Batlle, Ramón; Nerín, Cristina. 1999. Use of solid-phase microextraction for the analysis of bisphenol A and bisphenol A diglycidyl ether in food stimulants. Journal of Chromatography A. 864(1):137–144. Shao, Bing; Han, Hao; Hu, Jianying; Zhao, Jie; Wu, Guohua; Xue, Ying; Ma, Yalu; Zhang, Shujun. 2005. Determination of alkylphenol and bisphenol A in beverages using liquid chromatography/electrospray ionization tandem mass spectrometry. Analytica Chimica Acta. 530(2):245–252. Shao, Bing; Han, Hao; Tu, Xiaoming; Huang, Lei. 2007a. Analysis of alkylphenol and bisphenol A in eggs and milk by matrix solid phase dispersion extraction and liquid chromatography with tandem mass

Bisphenol A

spectrometry. Journal of Chromatography B. 850 (1–2):412–416. Shao, Bing; Han, Hao; Li, Dongmei; Ma, Yalu; Tu, Xiaoming; Wu, Yonging. 2007b. Analysis of alkylphenol and bisphenol A in meat by accelerated solvent extraction and liquid chromatography with tandem mass spectrometry. Food Chemistry. 105(3): 1236–1241. Stuart, James D.; Capulong, Christopher P.; Launer, Katherine D.; Pan, Xuejun. 2005. Analyses of phenolic endocrine disrupting chemicals in marine samples by both gas and liquid chromatography–mass spectrometry. Journal of Chromatography A. 1079(1–2): 136–145. Tavazzi, S.; Benfenati, E.; Barcelo, D. 2002. Accelerated solvent extraction then liquid chromatography coupled with mass spectrometry for determination of 4-octylphenol, 4-nonylphenols and bisphenol A in fish liver. Chromatographia. 56(7–8):463–467. Thomson, B.M.; Grounds, P.R. 2005. Bisphenol A in canned foods in New Zealand: An exposure assessment. Food Additives and Contaminants. 22(1): 65–72. Varelis, P.; Balafas, D. 2000. Preparation of 4,4′-(1-[2H6] methylethylidene)bis-[2,3,5,6-2H4]phenol and its application to the measurement of bisphenol A in beverages by stable isotope dilution mass spectrometry. Journal of Chromatography A. 883(1–2):163–170. Vom Saal, Frederick S.; Hughes, Claude. 2005. An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment. Environmental Health Perspectives. 113(8): 926–933. Watabe, Yoshiyuki; Kondo, Takuya; Imai, Hiroe; Masatoshi, Morita; Tanaka, Nobuo; Hosoya, Ken. 2004a. Reducing bisphenol A contamination from analytical procedures to determine ultralow levels in environmental samples using automated HPLC microanalysis. Analytical Chemistry. 76(1):105–109. Watabe, Yoshiyuki; Kondo, Takuya; Masatoshi, Morita; Tanaka, Nobuo; Haginaka, Jun; Hosoya, Ken. 2004b.

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Determination of bisphenol A in environmental water at ultra-low level by high-performance liquid chromatography with an effective on-line pretreatment device. Journal of Chromatography A. 103(1–2):45–49. Wen, Yi; Zhou, Bing S.; Xu, Ying; Jin, Shi W.; Feng, Yu Q. 2006. Analysis of estrogens in environmental waters using polymer monolith in-polyether ether ketone tube solid-phase microextraction combined with high-performance liquid chromatography. Journal of Chromatography A. 1133(1–2):21–28. Wetherill, Yelena B.; Petra, Christin E.; Monk, Kelly R.; Puga, A.; Knudsen, Karen E. 2002. The xenoestrogen bisphenol A induces inappropriate androgen receptor activation and mitogenesis in prostatic adenocarcinoma cells. Molecular Cancer Therapeutics. 1(1):515– 534. Ye, Xiaoyun; Kuklenyik, Zsuzsanna; Needham, Larry L.; Calafat, Antonio M. 2006. Measuring environmental phenols and chlorinated organic chemicals in breast milk using automated on-line column-switching– high performance liquid chromatography–isotope dilution tandem mass spectrometry. Journal of Chromatography B. 831(1–2):110–115. Yonekubo, Jun; Hayakawa, Kazuichi. 2008. Concentrations of bisphenol A, bisphenol A diglycidyl ether, and their derivatives in canned foods in Japanese markets. Journal of Agricultural and Food Chemistry. 56(6):2041–2047. Yoshida, Terumitsu; Horie, Masakazu; Hoshino, Youji; Nakazawa, Hiroyuki. 2001. Determination of bisphenol A in canned vegetables and fruit by high performance liquid chromatography. Food Additives and Contaminants. 18(1):69–75. Zoeller, R. Thomas; Bansal, Ruby; Parris, C. 2005. Bisphenol A, an environmental contaminant that acts as a thyroid hormone receptor antagonist in vitro, increases serum thyroxine, and alters rc3/neurogranin expression in the developing rat brain. Endocrinology. 146(2):2607–2612.

Chapter 16 Perfluoroalkylated Substances Leo M.L. Nollet

Definitions and health aspects Perfluorinated compounds (PFCs or PFASs) have been, and are used in a wide variety of industrial applications, such as textiles, paints, electronics, adhesives, and food packaging (Villagrasa et al. 2006; de Voogt and Saez 2006) because of their unique properties as repellents of dirt, water, and oils. Different abbreviations are used for perfluorinated compounds: • PS: perfluorinated surfactants • PFASs: perfluorinated and polyfluorinated alkyl substances • PFAAs: perfluorinated alkyl acids • PFCs: perfluorinated compounds • FASs: fluorinated alkyl substances As shown in Table 16.1 many PFASs exist, but two of them have been frequently studied: PFOA (perfluorooctanoic acid) and PFOS (perfluorooctane sulfonic acid) (Figure 16.1). PFASs are persistent in the environment, and some of them may be carcinogenic. For example, PFOA has been identified as a potent hepatocarcinogen in rodents (Andersen et al. 2008). The acute toxicity of PFASs is moderate, but some substances can induce peroxisome proliferation in rat livers and may change the fluidity of cell membranes (Jensen and Leffers Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

2008). Some of these PFASs, such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), are potential developmental toxicants and are suspected endocrine disruptors with effects on sex hormone levels resulting in lower testosterone levels and higher estradiol levels. Other PFASs have estrogenic effects in cell cultures. PFASs have been recognized as emerging contaminants in the food chain by the European Food Safety Authority (EFSA) (EFSA 2008; van Leeuwen and de Boer 2007). PFOSs and PFOAs are environmentally persistent and can be bioaccumulated in humans (Johansson et al. 2009). The European Food Safety Authority’s Panel on Contaminants in the Food Chain (CONTAM) evaluated these compounds regarding the importance of food and nonfood sources to human exposure. The indicative estimate of dietary exposure to PFOS is 60 and 200 ng/kg body weight (BW) per day for average and high consumers in Europe, respectively. For PFOA the indicative estimate of dietary exposure is 2 and 6 ng/kg BW per day for average and high consumers, respectively. Fish seems to be the major source of human exposure to PFOS and PFOA. Nonfood sources can contribute <2% and up to 50% for PFOS and PFOA, respectively. The panel established a tolerable daily intake (TDI) for PFOS of 150 ng/kg BW. For PFOA the panel established a TDI of 1.5 μg/ kg BW. The panel identified the need for 367

368

Analysis of Endocrine Disrupting Compounds in Food

Table 16.1. Full name, abbreviations, and chemical formula of a selection of PFASs. Full Name

Abbreviation

Poly- and perfluorinated acids Perfluorobutanoic acid Perfluorohexanoic acid Perfluoroheptanoic acid Perfluorooctanoic acid Perfluorononanoic acid Perfluorodecanoic acid Perfluoroundecanoic acid Perfluorododecanoic acid Perfluorotridecanoic acid Perfluorotetradecanoic acid Perfluoropentadecanoic acid

PFCAs PFBA PFHxA PFHpA PFOA PFNA PFDA PFUnA or PFUnDA PFDoA or PFDoDA PFTrA PFTA or PFTDA PFPA

Poly- and perfluorinated sulfonates Perfluorobutane sulfonate Perfluorohexane sulfonate Perfluorooctane sulfonate Perfluorodecane sulfonate Tetrahydroperfluorooctansulfonate

PFSAs PFBS or PFBuS PFHxS PFOS PFDS THPFOS

Non-ionic PFASs Perfluorosulfonamide N-ethyl perfluorooctane sulfonamidoethanol N-methyl perfluorooctane sulfonamidoethanol N-ethyl perfluorooctane sulfonamide

F

F

F

F

F

PFOSA NEtFOSE NMeFOSE NEtFOSA or NEtPFOSA

F

F

O

F F

F

F

F

F

F

OH

F

PFOA

F

F

F

F

F

F

F

F SO3H F F

F

F

F

F

F

F

F

PFOS Figure 16.1. PFOA and PFOS.

further data on the levels of these compounds in food and humans and recommended such investigations be promoted. The European Union has banned most uses of PFOS by EU directive 2006/122.

Chemical Formula CF3(CF2)2COOH CF3(CF2)4COOH CF3(CF2)5COOH CF3(CF2)6COOH CF3(CF2)7COOH CF3(CF2)8COOH CF3(CF2)9COOH CF3(CF2)10COOH CF3(CF2)11COOH CF3(CF2)12COOH CF3(CF2)13COOH CF3(CF2)3SO3− CF3(CF2)5SO3− CF3(CF2)7SO3− CF3(CF2)9SO3−

CF3(CF2)7SO2NH2 CF3(CF2)7SO2N(CH 2CH3)CH2CH2OH CF3(CF2)7SO2N(CH3) CH2CH2OH CF3(CF2)

Extraction and cleanup Figure 16.2 depicts extraction and cleanup methods for the analysis of PFASs in human and environmental matrices (van Leeuwen and de Boer 2007). After sample pretreatment consisting of protein precipitation and centrifugation, extractions are mostly performed by liquid– solid extraction (LSE), Soxhlet extraction, solid-phase extraction (SPE), and liquid– liquid extraction (LLE). Before the final determination a cleanup step may be necessary.

Determination Up to now, almost all analysis methods to determine PFASs have been based on liquid chromatography coupled to mass spectrometry or tandem mass spectrometry (LC–MS or LC–MS/MS) (Llorca et al. 2009; de Voogt and Saez 2006).

Cleanup

Extraction

Sample pretreatment

Sample-type

Perfluoroalkylated Substances

Sediment Soil Sludge* Precipitate*

Water Influent Effluent Precipitate

Biota

Whole blood Serum Plasma Milk

369

Air***

Filtration Centrifugation Filtration Centrifugation (Freeze)drying

PLE

LSE

Protein precipitation+ centrifugation

**

Soxhlet

Digestion (KOH) + SPE

LLE

SPE

SPE/XAD/PUF

Adsorption (C18/silica/ F-silica/graphitized carbon) and/or sulfuric acid Filtration

Final determination

* Soild phase ** Liquid phase *** Gaseous phase

Figure 16.2. Extraction and sample cleanup strategies for PFASs (from van Leeuwen and de Boer 2007).

Among them, the triple quadrupole (QqQ) MS is the most widely used analyzer because of its high dynamic range and good performance when working in selected reaction monitoring (SRM) mode (Villagrasa et al. 2006). In the recent LC–MS/MS methods, ion-paired, potassium hydroxide or solvent extractions were applied, for which the reported limits of quantification (LOQ) for PFOA and PFOS were as low as 1 μg kg−1 (de Voogt and Saez 2006). In Table 16.2 a selection of analysis methods of PFASs in different foodstuffs are given. The detection of all summarized methods are LC-MS/MS. The aim of the study of Llorca and others (2009) was the development and validation of a simple, sensitive, and selective analytical methodology to determine eight PFASs using

PLE (pressurized liquid extraction) with water and SPE on an ion exchanger for the extraction and preconcentration of PFASs from various fish samples, including liver, muscle, and roe. Analyzed compounds were perfluorobutane sulfonate (PFBS), perfluoropentanoic acid (PFPA), PFOA, PFOS, perfluoro-7-methyloctanoic acid (i,p-PFNA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoro-1decanesulfonate (L-PFDS). Analyte identification and confirmation was performed using an LC–QqLIT–MS/MS in compliance with the EU regulations (EU Commission Decision 2002/657/EC). Finally, PFAS residues were determined in different fishes taken in several markets of Valencia and Barcelona. An interlaboratory comparison was carried out among six laboratories for the

370 Ion pairing extraction SPE-Oasis-WAX method

SPE Oasis HLB cartridges 2 × 3 ml 2 : 2 : 1 methanol-acetoneethyl acetate SPE Waters Oasis 3 cc WAX cartridges 4 mL of 0.03% NH4OH in MeOH

Ion-pairing method (MTBE)

Chicken egg

Bluegill sunfish

Bluegill sunfish fillets

Drinking water, mussels, fish

PFOS PFHxS PFBS PFOSA PFDoDA PFUnDA PFDA PFNA PFOA PFHpA PFHxA PFHpA PFOA PFOS PFNA PFDA PFUnA PFDoA PFBS PFHxS PFOS 18O2-PFOS 13C2-PFOA PFHxA PFHpA PFOA PFNA PFDA PFUnA PFDoA PFHxS PFOS PFDS PFHpA PFOA PFUnDA

Sample Preparation

PFOS PFOA PFOSA PFHS PFOS

1 mL sample +3 mL formic acid sonication automated SPE Ion pairing extraction (MTBE)

Sample Milk Plasma (human and rat) Oysters

Analyte

Betasil C18 (2.1 × 100 mm, 5μm)

Phenomenex Luna C18 (2) (3 × 50 mm)

2 mM ammonium acetate and methanol

75% MeOH and 25% 2 mM ammonium acetate

ACN-water (0.1% acetic acid)

LC-ESIMS/MS

LC-ESIMS/MS

LC-ESIMS/MS

LC-ESIMS/MS

MeOH-water (2.0 mM ammonium acetate)

Hypersil Gold (2.1 × 100 mm, 3 μm)

LC-ESIMS/MS

ACN-water (2.0 mM ammonium acetate)

Detection LC-ESIMS/MS

Keystone Betasil C18 (2 × 50 mm) Keystone Betasil C18 (2.1 × 50 mm, 5 μm)

Mobile Phase MeOH-water (20 mM ammonium acetate)

Keystone Betasil C8 (3 × 50 mm)

Stationary Phase

Table 16.2. Analysis methods of PFASs in different foodstuffs. LOD-LOQ

For PFHxS, PFOS, PFOA, PFDS, and PFOSA: all less than 1 ng/g ww

0.08 ng/mL 0.01 0.01 0.08 0.05 0.01 0.01 0.05 0.01 0.01 0.01 0.5 ng/L 1 0.1 0.1 0.05 0.5 0.05

Milk: 0.3 ng/mL-NR 0.2 ng/mL-NR 0.7 ng/mL-NR 0.3 ng/mL-NR NR-10 ng/g ww

References

Quinete et al. 2009

Delinsky et al. 2009

Loos et al. 2007

Kannan et al. 2002d Wang et al. 2008

Kulenyik et al. 2004

371

PFBuS PFHxS PFOS PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA PFHpA PFOA PFNA PFDA PFUA PFOS 6 : 2 FTUCA 8 : 2 FTUCA 10 : 2 FTUCA MPFOS MPFOA PFOA PFOS

PFHxS PFOS PFDcS PFOSA PFHxA PFHpA PFOA PFNA PFDcA PFUnA PFDoA PFTriA PFTeA PFPeDA PFOA PFOS

Analyte

Liquid extraction, SPE, and additional cleanup with EnviCarb

Liquid extraction, solid-phase extraction

Trap column [Reprosil-Pur C18 AQ (3 mm × 33 mm, 5 μm)]

Various raw and cooked foodstuffs

Inuit traditional foods

Breast milk

5 mM ammonium formate in Barnstead Diamond water (18 MΩ-cm) (solution A) and a 1 : 1 (v/v) solution of acetonitrile/methanol (solvent B).

55% of 2 mM ammonium acetate (B, adjusted to pH 5 with acetic acid) and 45% of acetonitrile (B)

Reprosil-Pur basic C8 (2 × 150 mm, 5 μm)

2 mM ammonium acetate (eluent A) and methanol (eluent B) 2 mM ammonium acetate in methanol and 2 mM ammonium acetate in water

LC-ESIMS/MS

LC-ESIMS/MS

LC-MS/MS Turbo ion spray UPLC-ESMS/MS

Detection LC-HRMS or LC-MS/ MS

Mobile Phase Buffered (4 mM ammonium acetate) methanol and water

Genesis C18 (2.1 × 50 mm)

Gemini C18 column (2 × 50 mm) Acquity BEH C18 (2.1 × 50 mm, 1.7 μm)

Liquid extraction (MTBE)

Stationary Phase

Fish

Sample Preparation Discovery HS C18 column

Sample Fish

LOD-LOQ

— — 200 ng/L 20 ng/L

0.02 ng/g fw 0.25 0.20 0.25 0.10 0.12 0.10 0.08 0.08 0.08 0.08 0.10 0.15 0.30 1.5 μg kg−1 2 μg kg−1

References

Völkel et al. 2008

Ostertag et al. 2009

Nania et al. 2009 Ericson et al. 2009b

Berger et al. 2009

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Analysis of Endocrine Disrupting Compounds in Food

Table 16.3. HPLC conditions of interlaboratory study for the determination of PFASs in human serum and milk. Laboratory 1 HPLC/ESI-MS/ MS Serum/milk Methanolammonium acetate gradient Thermo Betasil C8 column (100 mm × 2.1 mm, 5 μm) API 4000

Laboratory 2

Laboratory 3

Laboratory 4

Laboratory 5

Laboratory 6

HPLC/ TIS-MS/MS Serum Methanolammonium acetate gradient Thermo Betasil C8 column (50 mm × 3 mm, 5 μm) API 4000

HPLC/TIS-MS/ MS Serum Acetonitrileammonium acetate gradient Thermo Betasil C8 column (100 mm × 2.1 mm, 5 μm)

HPLC/ ESI-MS/MS Serum/milk Methanolammonium acetate gradient ACE C18 column (50 mm × 2.1 mm, 3 μm) API 4000

HPLC/ESI-MS/ MS Serum/milk Methanolammonium acetate gradient Phenomenex Luna C8 column (50 mm × 2.1 mm, 3 μm) API 2000

UPLC/ESI-MS/MS

API 5000

Serum Methanolammonium acetate gradient Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm) Quatro Premier XE

ESI, electrospray ionization; TIS, turbo ion-spray. Source: Keller et al. 2009.

determination of perfluorinated alkyl acid concentrations in human serum and milk standard reference materials (Keller et al. 2009) (Table 16.3). Also in this study, all methods were based on liquid chromatography coupled to tandem mass spectrometry. PFAA concentration measurements agreed for serum SRM 1957 using the different analytical methods in six laboratories and for milk SRM 1954 in three laboratories. The concentrations in these SRMs are similar to the average concentrations measured in human serum and milk today. Most of the reported literature has focused on the analysis of anionic perfluorinated compounds such as PFOS and PFOA using ion pair extraction (IPE) or SPE and LC–MS/MS. These methods are not well suited to the analysis of neutral hydrophobic perfluorooctanesulfonamide compounds such as PFOSA, N-ethyl perfluorooctanesulfonamide (N-EtPFOSA), or N,N-diethyl perfluorooctanesulfonamide (N,N-Et2PFOSA) as they were developed for the analysis of anionic perfluorinated sulfonates and carboxylates (Tittlemier et al. 2005). Neutral perfluorooctanesulfonamides generally have low and variable recoveries, plus high method detection limits, when determined by IPE–LC–MS/MS (Olsen et al. 2003).

A method for the analysis of neutral perfluorooctanesulfonamides was developed for three related compounds (PFOSA, NEtPFOSA, and N,N-Et2PFOSA) in solid matrices, including food and biota samples. The method involved solvent extraction (SE) in combination with gas chromatography– positive chemical ionization mass spectrometry (GC-PCI-MS). Performance of the SE–GC–PCI-MS method in the analysis of PFOSA and N-EtPFOSA was compared to that of an IPE–LC–MS/MS method. The SE– GC–PCI-MS method was also used to obtain data on the presence of PFOSA, N-EtPFOSA, and N,N-Et2PFOSA in selected food samples.

PFASs in foods A number of reports on the occurrence of PFASs in food and drink are available (Loos et al. 2008; Ericson et al. 2008; Fromme et al. 2007; FSA 2006). Concentrations of 13 PFASs (PFBuS, PFHxS, PFOS, THPFOS, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, PFTDA, and PFOSA [see acronym definitions in Table 1.1]) were determined in municipal drinking water samples from 40 locations in Catalonia, Spain (Ericson et al. 2009a). PFOS and PFHxS were the PFASs

Table 16.4. Recent analysis techniques of PFCs Country/ Region

PFCs Analyzed

Foodstuffs Analyzed

Main Results

References

PFOS was found in all analyzed samples (range: 0.3–13.9 ng/g ww) PFOS was detected in 4 food items (1–10 ng/g ww). PFOA was only detected in potatoes (1 ng/g ww). Other 10 PFCs were also detected in potatoes The highest concentrations (ΣPFOSAs) were found in fast food, and the lowest one in fish burger Perfluorinated acids were detected in 9 of 54 composite samples analyzed. PFOS and PFOA were the most frequently detected Neither PFOS nor PFOA could be detected (LOD: 1.5 and 3 ng/g ww, respectively) PFOS was the most prominent PFC found. The detected levels of the remaining compounds were comparatively not significant PFOS (<1–58.9 ng/g ww) and PFUnDA (<1– 31.6 ng/g ww) were the predominant congeners Seven PFCs were found in cod; 2 in milk, potatoes, and beef; and 1 in leeks Only PFOS (24/36 samples, various foodstuffs), PFOA (2/36 samples, milk only), and PFHpA (2/36 samples, milk only) were detected PFOS (range of levels: 0.21 to 1.68 ng/g ww in raw and cooked samples. PFOSAs were only detected in scallops. All cooking methods reduced PFA levels PFHxS (1 food item), PFOS (8 food items), PFHxA (5 food items), and PFOA (1 food item) were the only detected PFCs

Gulkowska et al. 2006

China (2 cities)

9

Seven types of seafood

United Kingdom

15

Food samples from the 2004 Total Diet Study (TDS)

Canada

5 perflurooctanesulfonamides (PFOSAs)

1992–2004 Canadian TDS

Canada

12

Meat, fish, fast food, and some food items prepared in their packaging

Mediterranean Sea (Italy coast)

PFOS, PFOA

Filets of swordfish

Japan (various zones)

10

Blood and liver of farm animals

Asia (various seas)

9

Livers of skipjack tuna

Belgium (Flanders)

17

Vegetables, milk, meats, and cod

Spain (Catalonia)

11

Canada

17 (PFOSAs and PFAs)

Vegetables, fruits, pulses, cereals, fish and seafood, meats, eggs, milk and dairy products, oils and fats Raw and cooked (baked, boiled, fried) fillets of fish species

Spain (Catalonia)

11

Raw and cooked (grilled, fried) meats, unpackaged and packaged salmon and lettuce, and other food items

FSA 2006

Tittlemier et al. 2006 Tittlemier et al. 2007

Corsolini et al. 2008 Guruge et al. 2008

Hart et al. 2009 de Voogt and Saez 2006 Ericson et al. 2008

Del Gobbo et al. 2008

Ericson et al. 2009b

Source: Ericson et al. 2009b.

373

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Analysis of Endocrine Disrupting Compounds in Food

most frequently found at concentrations of 58.1 and 5.30 ng/L, respectively. Detection limits ranged between 0.02 (PFHxS) and 0.85 ng/L (PFOA). Human liver and milk samples from Catalonia, Spain, were analyzed by SPE and UPLC-MS (Kärrman et al. 2009). Six PFASs were detected in liver. PFOS had the highest mean concentration (26.6 ng/g ww). Mean levels ranged between 0.50 and 1.45 ng/g ww. Only PFOS and PFHxS were detected in human milk, with mean concentrations of 0.12 and 0.04 ng/mL, respectively. K. Senthilkumar and others (2007) determined PFASs (PFOS, PFOA, PFBS, PFHxS, PFOSA, and PFDoA) in river water, river sediment, liver of market fish, and liver of wildlife samples from Japan. Among fish, only jack mackerel showed PFOA and PFOS at 10 and 1.6 ng/g ww. Samples of drinking waters from the Rhine-Ruhr area were taken and analyzed for PS by SPE-HPLC-MS/MS (Skutlarek et al. 2006). The maximum concentration of all drinking water samples was at 598 ng/L. The major component was PFOA at 519 ng/L. Municipal drinking water samples and samples of bottle water in supermarkets were collected in Tarragona Province, Spain. PFC analyses were performed by HPLC-MS (Ericson et al. 2008). In tap water, PFOS and PFOA levels ranged between 0.39 and 0.87 ng/L and between 0.32 and 6.28 ng/L, respectively. PFHpA, PFHxS, and PFNA were also detected. PFC levels were significantly lower in bottled waters. PFOS couldn’t be detected in any sample. Eight pooled egg samples from eight locations in China were analyzed for 11 PFASs (Wang et al. 2008). Only the PFOS (ranging from 107 to <0.08 ng/g w/w) PFUnDA was detected in all egg samples. PFOA, PFDA, PFDoDA, and PFNA were detected in some samples. Loos and others (2007) detected PFHpA (range: 0.3–0.8 ng/L), PFOA (range: 1.0– 2.9 ng/L), PFOS (range: 6.2–9.7 ng/L), PFNA

(range: 0.3–0.7 ng/L), PFDA (range: 0.1– 0.3 ng/L), PFUnDA (range: 0.1–0.4 ng/L), and PFDoA (range: 0.1–2.8 ng/L) in tap water in the region of Lake Maggiore (Italy). Table 16.4 gives a summary of recent studies (from 2006 to 2010) on the concentrations of PFASs in food.

References Andersen M.E., Butenhoff J.L., Chang S.C., Farrar D.G., Kennedy G.L., Lau C., Olsen G.W., Seed J., Wallacekj K.B. Perfluoroalkyl acids and related chemistries— toxicokinetics and modes of action. Toxicol Sci. 102(1):3–14, 2008. Berger U., Glynn A., Holmström K.E., Berglund M., Halldin E., Ankarberg E.H., Törnkvist A. Fish consumption as a source of human exposure to perfluorinated alkyl substances in Sweden: Analysis of edible fish from Lake Vättern and the Baltic Sea. Chemosphere. 76(6):799–804, 2009. Corsolini S., Guerranti C., Perra G., Focardi S. Polybrominated diphenyl ethers, perfluorinated compounds and chlorinated pesticides in swordfish (Xiphias gladius) from the Mediterranean Sea. Environ Sci Technol. 42(12):4344–4349, 2008. Del Gobbo L., Tittlemier S., Diamond M., Pepper K., Tague B., Yeudall F., Vanderlinden L. Cooking decreases observed perfluorinated compound concentrations in fish. J Agric Food Chem. 56(16):7551– 7559, 2008. Delinsky A.D., Strynar M.J., Nakayama S.F., Varns J.L., Ye X., McCann P.J., Lindstrom A.B. Determination of ten perfluorinated compounds in bluegill sunfish (Lepomis macrochirus) fillets. Environ Res. 109(8): 975–984, 2009. de Voogt P., Saez M. Analytical chemistry of perfluoroalkylated substances. TrAC-Trends Anal Chem. 25(4):326–342, 2006. Ericson I., Domingo J.L., Nadal M., Bigas E., Llebaria X., van Bavel B., Lindström G. Levels of perfluorinated chemicals in municipal drinking water from Catalonia, Spain: Public health implications. Arch Environm Cont Tox. 57(4):631–638, 2009a. Ericson I., Nadal M., van Bavel B., Lindstrom G., Domingo J.L. Levels of perfluorochemicals in water samples from Catalonia, Spain: Is drinking water a significant contribution to human exposure? Environ Sci Pollut Res. 15(7):614–619, 2008. Ericson Jogsten I., Perello G., Llebaria X., Bigas E., Marti-Cid R., Kärrman A., Domingo J.L. Exposure to perfluorinated compounds in Catalonia, Spain, through consumption of various raw and cooked foodstuffs, including packaged food. Food Chem Toxicol. 47(7):1577–1583, 2009b. European Food Safety Authority (EFSA). Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts. EFSA J. 653(1), 2008.

Perfluoroalkylated Substances

European Union (EU). Directive 2006/122/EG. Available at: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri =OJ:L:2006:372:0032:0034:en:PDF. Food Standard Agency (FSA). Fluorinated chemicals: UK dietary intake. London: Food Standard Agency, 2006. Fromme H., Schlummer M., Moller A., Gruber L., Wolz G., Ungewiss J., Bohmer S., Dekant W., Mayer R., Liebl B., Twardella D. Exposure of an adult population to perfluorinated substances using duplicate diet portions and biomonitoring data. Environ Sci Technol. 41:7928, 2007. Gulkowska A., Jiang Q., Man K.S., Taniyasu S., Lam P.K.S., Yamashita N. Persistent perfluorinated acids in seafood collected from two cities of China. Environ Sci Technol. 40(12):3736–3741, 2006. Guruge K.S., Manage P.M., Yamanaka N., Miyazaki S., Taniyasu S., Yamashita N. Species-specific concentrations of perfluoroalkyl contaminants in farm and pet animals in Japan. Chemosphere. 73(1 Suppl):S210–S215, 2008. Available at http:// www.sciencedirect.com/science?_ob=ArticleURL&_ udi=B6V74 - 4SBYYCF - 6 & _user=10 & _rdoc=1 & _ fmt=&_orig=search&_sort=d&_docanchor=&view= c & _searchStrId=1108996551 & _rerunOrigin= scholar.google & _acct=C000050221 & _version= 1 & _ u r l Ve r s i o n = 0 & _ u s e r i d = 1 0 & m d 5 =d14852d916e6d88818a3478441bcde35-aff1. Hart K., Kannan A.K. Temporal trends (1992–2007) of perfluorinated chemicals in northern sea otters (Enhydra lutris kenyoni) from South-Central Alaska. Arch Environ Contam Toxicol. 56(3):607–614, 2009. Jensen A.A., Leffers H. Emerging endocrine disrupters: Perfluoroalkylated substances. Int J Androl. 31(2): 161–169, 2008. Johansson N., Benford D., Carere A., de Boer J., Dellatte E., De Voogt P., Di Domenico A., Heppner C.W., Schoeters G., Schrenk D. EFSA’s risk assessment on PFOS and PFOA in the food. Toxicol Lett. 189(Suppl 1):S42, 2009. Kannan K., Hansen K.J., Wade T.L., Giesy J.P. Perfluorooctane sulfonate in oysters, Crassostrea virginica , from the Gulf of Mexico and the Chesapeake Bay, USA. Arch Environ Contam Toxicol. 42(3): 313–318, 2002. Kärrman A., Domingo J.L., Llebaria X., Nadal M., Bigas E., van Bavel B., Lindström G. Biomonitoring perfluorinated compounds in Catalonia, Spain: Concentrations and trends in human liver and milk samples. Environm Science Poll Research. Published online May 21, 2009. Available at: http://www.springerlink.com/ content/r67rr4w70086lw13/. Keller J.M., Calafat A.M., Kato K., Ellefson M.E., Reagen W.K., Strynar M., O’Connell S., Butt C.M., Mabury S.A., Small J., Muir D.C.G., Leigh S.D., Schantz M.M. Determination of perfluorinated alkyl acid concentrations in human serum and milk standard reference materials. Anal Bioanal Chem. Published online on October 27, 2009. Available at: http:// www.springerlink.com/content/pr7l521242g10264/. Llorca M., Farré M., Picó Y., Barceló D. Development and validation of a pressurized liquid extraction

375

liquid chromatography–tandem mass spectrometry method for perfluorinated compounds determination in fish. J Chromatogr A. 1216(43):7195–7204, 2009. Loos R., Locoro G., Huber T., Wollgast J., Christoph E.H., De Jager A., Gawlik B.M., Hanke G., Umlauf G., Zaldivar J.M. Analysis of perfluorooctanoate (PFOA) and other perfluorinated compounds (PFCs) in the river Po watershed in Northern Italy. Chemosphere. 71(2):306–313, 2008. Loos R., Wollgast J., Huber T. Polar herbicides, pharmaceutical products, perfluorooctanesulfonate (PFOS), perfluorooctanoate (PFOA), and nonylphenol and its carboxyaltes and ethoxyaltes in surface and tap waters around Lake Maggiore in Northern Italy. Anal Bioanal Chem. 387:1469–1478, 2007. Nania V., Pellegrini G.E., Fabrizi L., Sesta G., De Sanctis P., Luchetti D., Di Pasquale M., Coni E. Monitoring of perfluorinated compounds in edible fish from the Mediterranean Sea. Food Chem. 115(3):951–957, 2009. Olsen G.W., Hansen K.J., Stevenson L.A., Burris J.M., Mandel J.H. Environ Sci Technol. 37:888, 2003. Ostertag S.K., Tague B.A., Humphries M.M., Titlemier S.A., Man Chang H. Estimated dietary exposure to fluorinated compounds from traditional foods among Inuit in Nunavut, Canada. Chemosphere. 75(9):1165– 1172, 2009. Quinete N., Wu Q., Zhang T., Hun Yun S., Moreira I., Kannan K. Specific profiles of perfluorinated compounds in surface and drinking waters and accumulation in mussels, fish, and dolphins from southeastern Brazil. Chemosphere. 77(6):863–869, 2009. Senthilkumar K., Ohi E., Sajawan K., Takasuga T., Kannan K. Perfluorianted compounds in river water, river sediment, market fish, and wildlife samples from Japan. Bull Envrion Contam and Tox. 79(4):427–431, 2007. Skutlarek, D., Exner M., Fárber H. Perfluorinated surfactants in surface and drinking waters. Environ Sci Poll Res. 13(5):299–307, 2006. Tittlemier S.A., Pepper K., Edwards L. Concentrations of perfluorooctanesulfonamides in Canadian total diet study composite food samples collected between 1992 and 2004. J Agric Food Chem. 54(21):8385–8389, 2006. Tittlemier S.A., Pepper K., Edwards L., Tomy G. Development and characterization of a solvent extraction–gas chromatographic/mass spectrometric method for the analysis of perfluorooctanesulfonamide compounds in solid matrices. J Chromatogr A. 1066(1–2):189–195, 2005. Tittlemier S.A., Pepper K., Seymour C., Moisey J., Bronson R., Cao X. -L. Dabeka R.W. Dietary exposure of Canadians to perfluorinated carboxylates and perfluorooctane sulfonate via consumption of meat, fish, fast foods, and food items prepared in their packaging. J Agric Food Chem. 55(8):3203–3210, 2007. Also available at: http://pubs.acs.org/doi/abs/10.1021/ jf0634045-jf0634045AF2. Van Leeuwen S.P.J., de Boer J. Extraction and clean-up strategies for the analysis of poly- and perfluoroalkyl

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substances in environmental and human matrices. J Chromatogr A. 1153(1–2):172–185, 2007. Villagrasa M., de Alda M.L., Barcelo D. Environmental analysis of fluorinated alkyl substances by liquid chromatography–(tandem) mass spectrometry: A review. Anal Bioanal Chem. 386(4):953–972, 2006. Völkel W., Genzel-Boroviczény O., Demmelmair H., Gebauer C., Koletzko B., Twardella D., Raab U., Fromme H. Perfluorooctane sulphonate (PFOS) and

perfluorooctanoic acid (PFOA) in human breast milk: Results of a pilot study. Int J Hyg Environ Health. 211(3–4):440–446, 2008. Wang Y., Yeung L.W.Y., Yamashita N., Taniyasu S., So M.K., Murphy M.B., Lam P.P.S. Perfluorooctane sulfonate (PFOS) and related fluorochemicals in chicken egg in China. Chin Sci Bull. 53(4):501–507, 2008.

Chapter 17 Flame Retardants D. Lambropoulou, E. Evgenidou, Ch. Christophoridis, E. Bizani, and K. Fytianos

Introduction Flame retardants (FRs) comprise a diverse group of chemicals that can be added to or applied as a treatment to all sorts of flammable material, such as plastics, wood, textiles, paper, natural fiber, building materials, etc. Alternatively, they can be used during the production process as a chemical modification of some plastic materials. The use of FRs is primarily to protect materials against ignition and to prevent fire-related damage (EFRA). They are incorporated into products in two ways; either as additive or reactive FRs. Additive FRs are mixed into the product at manufacture, whereas reactive flame retardants are bound into the polymer framework during manufacture. The former are more likely to leach from finished consumer products than the latter. The annual consumption of FRs is currently over 1.5 million tons (MSFR 2008). Many types of FRs are commercially available (over 200 different substances), including alumina trihydrate, magnesium hydroxide, ammonium polyphosphate, halogenated (chlorinated and brominated) and organophosphorus compounds, and nitrogen-containing materials. The choice of FR depends on economic considerations, the polymer matrix involved (compatibility is required between the FR and the polymer),

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

the temperature at which it is to be used, and the level of fire protection desired. Currently, because of their high performance efficiency and low cost, the most widely encountered FR materials are the brominated flame retardants (BFRs), and this family of compounds is discussed in this chapter based on their high production and consumption as well as on their occurrence in different food matrices. These compounds are found in a wide variety of materials including paints, plastics, textiles, furniture, and electronics and may be either covalently bonded to the polymer or additively mixed into the final product. Furthermore, BFRs are routinely included in the manufacture of household goods, and an increasing consumer demand for such products has been reflected in the global BFR production patterns over the past several years (Arias 2001; Ward et al. 2008). The BFRs that have been shown to be environmentally persistent and bioaccumulative include polybrominated diphenyl ethers (PBDEs; 209 separate congeners), hexabromocyclododecane (HBCD) isomers (3 diastereomers α, β, and γ), and to a lesser extent polybrominated biphenyls (PBBs; also 209 congeners) and tetrabromobisphenol A (TBBP-A). Despite the ban on usage of PBBs (Hites 2006) and some PBDEs (penta-BDE and octa-BDE in the European Union since August 2004 and deca-BDE, since July 2008) (European Union 2008, Directive 2003/11/ EC), the production of some of these compounds continues to be generally high, and it 377

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Analysis of Endocrine Disrupting Compounds in Food

is estimated that more than 200,000 tons of BFRs are produced globally each year (Birnbaum and Staskal 2004). For example, the global consumption estimates of TBBPA, which was reported as the BFR with the highest production volume, covering about 60% of the total BFR market, vary from 120,000 to 150,000 tons/year, including TBBP-A derivatives (BSEF 2009; Covaci et al. 2009a). HBCD is the third most widely used BFR (accounting for 8.2% of total market demand in 2001) and is in second place in the European Union. Furthermore, worldwide, more than 70,000 metric tons of PBDEs have been produced annually, half of which have been used in the United States and Canada, including almost all of the pentaPBDE manufactured (Hites 2006). Globally, high levels of BFRs have been detected in a variety of matrices, including biota, sediments, air, water, marine mammals, and even in human breast milk (Hale et al. 2006; Law et al. 2006, 2008; Covaci et al. 2007a, 2009a; Yogui and Sericano 2009; Eljarrat and Barceló 2009). The consumption of contaminated food (particularly fish) is thought to be a major pathway to human exposure. In a recent meta-analysis of global concentration data, Hites (2004) determined that human PBDE levels had increased by a factor of 100 over the past three decades, with concentration loads doubling every 5 years. Furthermore, in a more recent report it was demonstrated that North Americans have the highest global body burdens, averaging contaminant levels 20 times higher than that of Europeans (Ward et al. 2008; Rayne 2009). The widespread environmental distribution of BFRs is well-known, and a number of studies published in recent years stress the continuing interest and high level of activity in research on the analysis, environmental distribution, fate, transport, and effects of BFRs. This chapter gives an overview on recent trends in sample preparation and detection for the determination of BFRs in food, including some key applications. Results of food-monitoring programs, highlighting the

occurrence of BFRs in fish, meat, eggs, and dairy products, are also presented.

Emission, transformation, and distribution of brominated flame retardants in the environment Any strategies to limit exposure to BFRs require an understanding of their sources and fate in the environment. The most common urban sources for emissions of BFRs are releases during their use in manufacturing commercial products and releases from commercial products such as furniture, electronic equipment (e.g., computers, televisions), and small motor appliances (e.g., hair dryers) during their use and subsequent disposal. As far as emissions related to in-use products are concerned, BFRs are emitted indoors and then move outdoors. Thus, atmospheric deposition is the major source of BFRs to many environmental compartments. Recycling and/ or disposal of wastes containing BFRs, manufacturing output, fluvial deposition from tributaries and wastewater treatment plants, and incineration of materials containing PBBs and PBDEs, as well as during accidental fires, are generally considered as other sources. The properties of individual BFR compounds vary, and their environmental fate depends on the properties of each specific compound (Table 17.1) (Cousins and Palm 2003). For example, the release and environmental transport of PBDEs, as semivolatile and hydrophobic compounds, is presumably analogous to that of polychlorinated biphenyls (PCBs). Similar to the PCBs, in that they generally have limited biodegradability, they are expected to accumulate in organic-rich media such as soils, sediments, and lipid-rich biotic tissues and to biomagnify in food chains. PBDEs, however, are less volatile and less water soluble at the same level of halogenation and thus partition more strongly to atmospheric aerosols, soil, and sediment particles. As a result, they will demonstrate lower bioavailability and are less mobile in the environment than PCBs. The fate processes also

Table 17.1. Summary of measured physical-chemical properties of the main PBDE congeners, HBCDs and TBBP-A. BFRs

Structure

Polybrominated diphenyl ethers PBDEs

CAS No.

Log Kow

Water Solubility at 25°C (mg L−1)

Henry’s Constant (atm m3 mol−1)

4131875-6

5.47– 5.58

0.00038

—

543643-1

5.87– 6.55

0.00007 0.015 0.0496 0.0947

1.4805 × 10−5 1.0926 × 10−5

6034860-9

7.32

0.0109

2.26992 × 10−6

18908464-8

7.24

0.009

6.80977 × 10−7

6863149-2

7.90

0.04

6.61238 × 10−7

20712215-4

6.86– 7.93

4.08 × 10−6 8.70 × 10−7

2.3688 × 10−6

116319-5

12.11

1.00 × 10−4

1.62 × 10−6 1.93 × 10−8 1.2 × 10−8 4.4 × 10−8

79-94-7

4.5 5.3

1.00 × 10−3

7.05 × 10−11

O

Brx x + y = 1–10 Bry 2,4,4′-Tribromo diphenyl ether (BDE-28)a

Br Br

2,2′,4,4′-Tetrabromo diphenyl ether (BDE-47)a

O

Br

Br

Br O Br

2,2′,4,4′,5-Pentabromo diphenyl ether (BDE-99)b

Br Br

Br

O

Br

Br

2,2′,4,4′,6-Pentabromo diphenyl ether (BDE-100)b

Br

Br O

Br 2,2′,4,4′,5,5′-Hexabromo diphenyl ether (BDE-153)b

Br

Br

Br Br

Br

O

Br

Br

Br Br

2,2′,4,4′,5,6′-Hexabromo diphenyl ether (BDE-154)a

Br Br Br

Br O

Br Decabrominated diphenyl ether (BDE 209)c

Br Br

Br O

Br Br

Br Br

Br

Br Br Br

H3C CH3

Tetrabromobisphenol-Ac Br

Br

HO

OH Br

Br (continued)

379

380

Analysis of Endocrine Disrupting Compounds in Food

Table 17.1. Summary of measured physical-chemical properties of the main PBDE congeners, HBCDs and TBBP-A. (cont.) BFRs

Structure

Hexabromocyclododecanes HBCDs (3 diastereoisomers)d

Br Br

CAS No.

Log Kow

Water Solubility at 25°C (mg L−1)

Henry’s Constant (atm m3 mol−1)

2563799-4

5.6

13423750-6

—

48.8 × 10−3

—

13423751-7

—

14.7 × 10−3

—

13423752-8

—

2.1 × 10−3

—

Br Br

Br Br

α-Hexabromocyclododecanes α-HBCDd

Br

Br Br Br

β-Hexabromocyclododecanes β-HBCDd

Br

Br

Br

Br Br Br

γ-Hexabromocyclododecanes γ-HBCDd

Br

Br

Br

Br Br Br

Br

Br

a

Mackay D., Yin S.W., Ma K.-C., Chi L.S. 2006. Handbook of Physical-Chemical Properties and Environmental Fate for Organic Chemicals, Vol. III. Oxygen Containing Compounds. CRC Press Taylor & Francis Group, Boca Raton, FL. b Public health statements for PBDEs. Agency for Toxic Substances and Disease Registry. Available at www.atsdr.cdc.gov/phs/ phs.asp?id=899&tid=183. Accessed June 5, 2010. c United States National Library of Medicine. ChemIDplus Advanced Database. Available at http://chem.sis.nlm.nih.gov/ chemidplus/. Accessed June 5, 2010. d Covaci A., Gerecke, A.C., Law R.J., Voorspoels S., Kohler M., Heeb N.V., Leslie H., Allchin C.R., de Boer J. 2006. Hexabromocyclododecanes (HBCDs) in the environment and humans: A review. Environ. Sci. Technol. 40(12):3679–3688.

include regional and long-range transport, evidenced by the fact that they have been detected in the Arctic (de Wit et al. 2006). Recent studies regarding the fate of PBDEs have shown that PBDEs can be debrominated to less-brominated PBDE congeners, or they can form chlorinated dibenzofurans photolytically by both UV light, and under certain conditions, by natural sunlight, with half-lives

ranging from 15 min up to 81 h (Rayne et al. 2003; de Wit 2002). Several studies have shown an inverse relationship between potential toxicity and number of bromine atoms among the BDE congeners (Meerts et al. 2001). Therefore, debromination of BDE 209 (decabromodiphenyl ether) in the environment can lead to potentially more toxic degradation products. Finally, there are studies

Flame Retardants

that indicate that during thermal stress, PBDEs and TBBP-A are converted to the dioxin-like compounds, polybrominated dibenzodioxins (PBDDs) and polybrominated dibenzofurans (PBDFs) (Janssen 2005). Like PBDEs, HBCD is lipophilic and very persistent in the environment. Based on its physicochemical properties, a moderate potential for long-range transport of HBCD has been estimated. Like other BFRs, the estimated maximum distance is approximately 2600 km in both air and water. Consistent with the high hydrophobicity, considerable bioconcentration has been reported for HBCD (log BCF = 4). Biotransformation occurs predominantly under anaerobic conditions, with half-lives ranging from approximately 2 days to 2 months (Davis et al. 2005). Complete debromination by dehaloelimination has been confirmed for all diastereomers of the technical product (Davis et al. 2006). The partitioning of TBBP-A in the environment and biota differs from that of PBDEs and HBCDs. For example, at neutral pH, TBBP-A has very low solubility and its soil mobility is expected to be minimal. However, at higher pH (as in some arid soils areas), the solubility of TBBP-A increases, making its soil mobility and potential for groundwater contamination considerable (Hakk and Letcher 2003). Bioaccumulation of TBBP-A in fatty tissues and in the food chain is minimal. Similarly to PBDEs, TBBP-A is photolytically decomposed when exposed to UV light. The breakdown products correspond to different brominated organic compounds such as dibromophenol, 2,4,6tribromophenol, and di- and tribromobisphenol A (de Wit 2002). Reductive debromination of TBBPA to bisphenol-A (BPA), a known estrogenic compound and suspected teratogen, under anaerobic conditions was also reported (Ronen and Abeliovich 2000). Finally, biotransformation reactions besides biodegradation in the environment gives rise to a dimethylated derivative of TBBP-A, which is highly lipophilic and thus more bio-

381

accumulable than TBBP-A (Allard et al. 1987).

Toxicity of flame retardants The fact that PBDEs can accumulate in living organisms has raised concerns about their toxic potential. There is no clear evidence yet for PBDEs being a hazard for either humans or wildlife. Unlike dioxins and PCBs, PBDEs are not strong inducers of the aryl hydrocarbon hydroxylase (Ah) receptor. However, some recent laboratory studies, including a few on BDE-209, have shown potential effects on the nervous system, immune system, and hormones (Darnerud 2003; Mariussen and Fonnum 2003, 2006). Moreover, the presence of PBDEs in human tissue is of a particular concern due to their possible association with endocrine disruption, reproductive (Legler 2008) and/or developmental toxicity (including neurotoxicity), and cancer in rodent studies (Schecter et al. 2004). The penta- and octa-BDE mixtures are now prohibited in the European Union due to their potential as environmental toxicants. In recent years, the main use of penta-BDE has thus been in North America. This may be the reason why PBDEs are found at an order of magnitude higher concentrations in mother ’s milk and blood samples collected from U.S. residents compared to Europeans (Schecter et al. 2003; Hites 2004). Both the penta- and the octa-BDE mixtures are currently under consideration to be classified and included as persistent organic pollutants (POPs) under the Stockholm Convention. The toxicological database for HBCDs is still limited. Acute toxic effects appear to be low (Darnerud 2003). However, there are indications that oral exposure to HBCDs induces drug-metabolizing enzymes in rats, such as hepatic cytochrome P450 (CYP) (Germer et al. 2006), and that HBCDs may induce cancer by a nonmutagenic mechanism (Helleday et al. 1999). There are reports that HBCDs can disrupt the thyroid hormone

382

Analysis of Endocrine Disrupting Compounds in Food

system (Danrerud 2003) and affect the thyroid hormone receptor–mediated gene expression (Yamada-Okabe et al. 2005). Following neonatal exposure experiments in rats, developmental neurotoxic effects can be induced, such as aberrations in spontaneous behavior, learning, and memory function (Eriksson et al. 2002). HBCDs can also alter the normal uptake of neurotransmitters in the rat brain (Mariussen and Fonnum 2003). Oral administration studies with rats and mice indicate that TBBP-A has a low acute toxicity. Due to the structural resemblance to the thyroid hormone thyroxin (T4) and bisphenol A, a suspected endocrine disruptor, the major concern regarding TBBP-A is its potential as an endocrine disruptor. Studies have shown that TBBP-A acts both as a thyroid hormone and estrogen agonist (Kitamura et al. 2002). Moreover, TBBP-A proved to be a rather potent inhibitor of the sulfation of estradiol by estrogen sulfotransferase (Kester et al. 2002). Furthermore, TBBP-A was shown to be a potent in vitro inhibitor for the binding of T4 to transthyretin, the thyroid hormone–binding transport protein in plasma (Hamers et al. 2006). TBBP-A is also immunotoxic and appears to interfere with cellular signaling pathways (Strack et al. 2007). Excellent reviews of recent studies demonstrating the endocrine disrupting (ED) effects of BFRs and other toxicological aspects have been published and should be consulted for further details (Legler 2008; Talsness 2008).

Analytical methodologies Currently, The analysis of BFRs in various food and feed samples is a problem of primary concern for quality control laboratories due to human and animal health risks associated with the accumulation of these substances. In 2003, the European Community (EC) introduced a new regulation to control the presence of PBDEs in the environment (Directive

2003/11/EC), with the intention of introducing new regulations similar to those existing for PCDD/Fs and PCBs for foodstuffs (EC/ 2375/2001; ESR 1993) in the near future. Additionally, in 2006, the European Food Safety Authority (EFRA) has recognized the concern for the contamination of food and feed with BFRs and has adopted an opinion in which the monitoring of BDE 28, 47, 99, 100, 153, 154, 183, 209; BB-153; and HBCD is recommended (EFSA 2006). These directives and legislative implications involve routine monitoring, calling for adaptation of analytical methods in order to provide adequate sensitivity and selectivity so as to allow unambiguous determination of BFRs in complex matrices (Gómara et al. 2006a). Several reviews have been published in recent years that deal with the analytical procedures to determine the presence of BFRs in environmental, biological, and human samples (de Boer and Wells 2006; Covaci et al. 2006, 2007a, 2008, 2009a). Generally speaking, the analysis of BFRs demands the use of complicated, time- and labor-consuming analytical procedures, and high analytical skills. This is due to both the inherent complexity of the matrix and the requirement of low limits of detection (LODs). Moreover, the analytical methodologies are especially difficult due to the complexity of the mixtures of congeners (209 PBDEs, etc.). The different toxicity of each congener requires the development of congener-specific methods. In the 1990s, only a small number of laboratories had the necessary expertise to determine different classes of BFRs. Worldwide, several laboratories have recently started to develop PBDE analysis methods. An even smaller, but growing number of laboratories have started to develop analytical methods for BDE-209, HBCD, and TBBP-A. Following the general regulations that come from EC directives, a series of worldwide interlaboratory studies was organized to assist these laboratories in their method development (de Boer and Wells 2006). In general, most analytical strategies

Flame Retardants

adopted by official and research laboratories include several analytical steps, depending on the complexity of the matrix, with sample pretreatment, extraction cleanup and fractionation, and quality control (QC) and BFR detection as the common sequence. Sample preparation strategies, cleanup and fractionation, injection techniques, chromatographic separation, and detection methods are briefly described in the next section. Comprehensive overviews of the analytical methodologies and detailed descriptions of the influence and extent of matrix effects in quantitative BFR analysis using gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) are provided in reviews and textbooks cited and should be consulted for detailed analytical planning and better understanding of the problems linked to inconstant ionization and matrix effects (de Boer and Wells 2006; Covaci et al. 2007a, 2008, 2009a).

Sample preparation The physical-chemical properties of PBDEs are very similar to those of PCBs or other POPs, and in general, methods applied for FR analysis to food matrices are simple adaptations of those used for the analysis of other POPs. Pretreatment steps, including homogenization and drying of the sample, which can be performed by air-drying, freeze-drying, or drying by mixing the sample with sodium sulfate, are usually carried out before extraction of food samples to enhance the extraction’s performance. Examples of various approaches commonly used for determining BFRs in food matrices are given in Table 17.2. Sample preparation is focused on commonly used techniques, such as solvent extraction (SE), Soxhlet extraction, hot Soxhlet extraction, ultrasonic extraction (USE), supercritical fluid extraction (SFE), accelerated solvent extraction (ASE), microwave-assisted extraction (MAE), and matrix solid-phase dispersion (MSPD). So

383

far, the most commonly applied approach for BFR extraction in food matrices (especially for fish samples) is Soxhlet. In general, extraction with a polar-nonpolar binary mixture in different proportions (either 1 : 1 or 1 : 3, v/v) has been found to be efficient for recovering BFRs in complex biological tissues and fatty foodstuffs. Binary mixtures such as acetone-hexane (Morris et al. 2006; Guo et al. 2007; van Leeuwen and de Boer 2008; Wan et al. 2008) and DCM-hexane (Sajwan et al. 2008; Hajslova et al. 2007) appear to be the most commonly used for the extraction of fish, shellfish, and marine species. Soxhlet and n-hexane-acetone (3 : 1, v/v) were also used to extract PBDEs from various food items, including hamburgers and pizzas (Voorspoels et al. 2007). The Soxhlet extraction times (6–24 h) can be substantially shortened by replacing the traditional Soxhlet system with a semiautomated hot Soxhlet. The use of hot Soxhlet allows a significant reduction in the extraction time without affecting the final results. High recoveries (82%–93%, RSDs <16%) have been achieved after 2 or 2.5 h hot Soxhlet extraction with n-hexane/acetone (3 : 1, v/v) of chicken eggs (Covaci et al. 2009b) and fish tissues (Roosens et al. 2008) for PBDE and simultaneous analysis of PBDEs and HBCDs, respectively. A number of disadvantages have been noted with Soxhlet methods: they are laborious, time-consuming, and subject to problems arising from the evaporation of large volumes of solvent and the disposal of toxic or flammable solvents. To overcome these drawbacks, new trends in the sample treatment and miniaturization have resulted in new extraction techniques that use much smaller amounts of organic solvent, provide superior extraction efficiencies, permit the on-line coupling to analytical measurement techniques, and allow easier automation and higher extraction throughput. Interesting examples in this area include the new generation of enhanced extraction technologies such as pressurized liquid extraction, or PLE (also

384

Fish/vegetable oil Fish

Milk

PBDEs (7)

PBDEs (30) TBBP-A

Chicken eggs Fish fillets Fish (salmon)

Marine species

Fish & marine species Fish (marine and freshwater) Fish

HBCD PBCDE PBDEs (9)

PBDEs (16) HBCD TBBP-A Me-TBBP-A PBDEs (13)

PBDEs (4) TBBP-A

PBDEs (43)

Caviar

Homogenization, freeze-drying Homogenization, freeze-drying Homogenization + Na2SO4 Homogenization + Na2SO4

Fish

PBDEs (10) HBCD

PBDEs (22)

Homogenization

Fish

PBDEs (7)

Homogenization + Na2SO4 + sea sand

Shellfish

Homogenization, freeze-drying Homogenization, freeze-drying Homogenization + Na2SO4 (12 h)

Freezedrying + reconstituting with H2O Freeze-drying

—

Pretreatment

PBDEs (15)

HBCDs

PBDEs (6)

Sample Type

BFRs (no. of congeners)

GPC

Soxhlet (8 h, hex-DCM [1 : 1])

Column extraction (ethyl acetate/petroleum ether, 1 : 10)

Soxhlet, (DCM 18–24 h)

Soxhlet, (18 h, Acet/DCM, 1 : 1)

Soxhlet, (24 h, DCM/hex, 3 : 1)

Column extraction (hex/acet, 2 : 1) Soxhlet (7 h, diethyl ether/hex [3 : 1]) Soxhlet, (hex/acet, 3 : 1) + acidified water

Soxhlet (17 h, DCM/hex, 3 : 1)

H2SO4/Na2SO4/activated Cu/ activated SiO2 GPC + Florisil + alumina column GPC + Florisil column + H2SO4

Acidified silica / Na2SO4 column

GPC + LLE with H2SO4

GPC + activated Florisil column

GPC + SiO2

Fractionation on SiO2 (2 g), elution with DCM/hex (1 : 4) Hex layer (PBDEs): Oasis HLB SPE + SiO2+SiO2–H2SO4 ACN layer (HBCD + TBBP-A): enzymatic hydrolysis (501C, 4 h) + Oasis HLB + SiO2 SPE GPC + multilayer SiO2/alumina column Florisil + SiO2 column

MSPD (Na2SO4, 2 g + Florisil, 1.5 g) + SiO2–H2SO4 (C6, 20 mL) SE (AcN + hex)

Soxhlet (48 h, Acet)

SiO2 + H2SO4

Cleanup/Fractionation

USE (hex. 5 min)

Extraction Procedure

Table 17.2. Analytical procedures used for the determination of PBDEs, HBCDs, and TBBP-A in selected matrices.

GC-MS

HRGC-MS

GC-MS GCxGCTOFMS GC/ECD GC/EI-MS GC-ECD GC-MSD GC-ECNIMS LC-ESI-MS/ MS GC-ENCI/ MS GC-MS

GC-ECD

LC-ESI-MS/ MS GC-NCI-MS

GC-EIHRMS

GC-ECNIMS GC-ECD

Instrument

>95

—

76–108

73–84

—

70–110

>90

86–103

100

83–101

40–110

81–106

92–101

Rec. (%)

Hiebl and Vetter 2007 Shaw et al. 2008 van Leeuwen and de Boer 2008 Wan et al. 2008 Cheung et al. 2008 Staskal et al. 2008 Wang et al. 2008

Guo et al. 2007 Sajwan et al. 2007 Hajslova et al. 2007

Cariou et al. 2005

Jacobs et al. 2004 Martinez et al. 2005

References

385

Fish, mussels

Fish

PBDEs

PBDEs (35) HBCD PBDEs (6) HBCD

Seafood

Fish

Fish, mussels

PBDEs (8)

PBDEs (8)

Homogenization, lyophilization

Homogenization + Na2SO4 Homogenization + Na2SO4 Freeze-drying

Homogenization + Na2SO4 Homogenization

Freeze-drying

Homogenization (composite samples)

Pretreatment

ASE, (100°C, 500 psi, hex/ DCM, 1 : 1) MAE (hex, 400 W)

SE (hex) + blended with acidified SiO2 + column extraction (multilayer SiO2-Na2SO4 column + carbon column + H2SO4) (ASE, 100 0C, 1500 psi) (acet/ DCM (1 : 3, v/v). Column extraction (cyclohex/ DCM) SE (potassium oxalate solution, ethanol, diethyl ether, and n-pentane) Hot Soxhlet, (2.5 h, hex/acet, 3 : 1) Hot Soxhlet, (2 h, hex/ acet, 3 : 1) ASE

Extraction Procedure

Acet, acetone; AcN, acetonitrile; Hex, n-hexane; DCM, dichloromethane. Solvent mixtures: proportions as v/v.

Fish

Chicken eggs

PBDEs (13) HBCDs PBDEs (11) HBCD PBDEs (10)

Whitefish

Shellfish

Sample Type

PBDEs (17)

BFRs (no. of congeners)

SPE, hex

Multi layer (acidified–modified SiO2/AgNO3//Na2SO4) + activated basic Al3O4 + HPLC carbon column H2SO4

Acidified SiO2

69–87 78–100

GC-μECD

116–135

65–90

60–95

23–62

60–120

80–95

—

Rec. (%)

GC/NCI-MS

GC-MS LC-MS/MS HRGC– HRMS

GC-MS

GC-ECD GC-EI/MS HRGC/ HRMS HRGC/ HRMS

GPC +SiO2 + 5% H2Odeactivated SiO2 Acidified SiO2/Al2O3 H2SO4 + GPC + acidified SiO2 + basic Al3O4 + carbon column Acidified SiO2

GC-HRMS

Instrument

Acid modified SiO2 / base modified SiO2 + activated Al3O4

Cleanup/Fractionation

Shelver et al. 2008 Fajar et al. 2008

Covaci et al. 2009b Roosens et al. 2008 Miyake et al. 2008

Mizukawa et al. 2009 Shaw et al. 2009 Bogdal et al. 2009

Fernandes et al. 2008

References

386

Analysis of Endocrine Disrupting Compounds in Food

known as accelerated solvent extraction, or ASE), microwave-assisted extraction (MAE), and supercritical fluid extraction (SFE). The enhanced efficiency of these methods derives from elevated solvent temperature, which speeds up extraction of analytes from solid matrices as a result of increased solubilities, better desorptions, and enhanced diffusion. All of these methodologies have been successfully employed for the determination of BFR compounds in food solid and semisolid matrices, with PLE being preferable. Applicability of PLE to determine BFRs in different food matrices has been successfully proved by many authors. For example, Mizukawa et al. (2009) used the PLE method (100°C, 1500 psi, acetone/DCM, 1 : 3, v/v) to determine the biomagnification of 20 PBDE congeners in lower-trophic-level organisms. Miyake et al. (2008) also applied highresolution gas chromatography/high-resolution mass spectrometry (HRGC-HRMS), after a preconcentration carried out with PLE, to determine PBDEs and polybrominated dibenzo-p-dioxins, dibenzofurans (PBDDs/ DFs) in common seafood. PBDE recoveries obtained using the PLE method after cleanup with column chromatography and fractionation were between 116% and 135% for the tested PBDE congeners (RSD <40%). PLE is increasingly being used to replace Soxhlet and column extraction methods. The use of PLE-based extraction methods for organic pollutants, including BFRs, has recently been reviewed by Bjorklund et al. (2006). Apart from PLE, MAE has attracted growing interest for BFR analysis because it allows rapid extraction of solutes from solid matrices by employing microwave energy as a source of heat, with extraction efficiency comparable to that of the classical techniques. Although MAE is now established as a routine, well-developed method for sample preparation in environmental analysis (soils, sediments, domestic dust, etc.) (Regueiro et al. 2006, 2007), electronic materials (Li et al. 2009; Vilaplana et al. 2009), and sty-

renic polymers (Vilaplana et al. 2008), few instances have been found in the literature for the application of MAE to the extraction of FRs from solid foodstuffs (Bayen et al. 2004; Carro et al. 2007). Recoveries obtained from spiked marine biological tissues with variable fat and moisture contents (range 1.2%–38% and 48%–78%, respectively) using the MAE method for three PBDE congeners (BDEs 47, 99, and 100) were in the range of 89%–97%. Comparing MAE to Soxhlet extraction by analyzing two certified reference materials (CRMs) (SRM 1588a, cod liver oil; SRM 2978, mussel tissue), insignificant differences in concentration levels were obtained. Furthermore, the advantage of reducing solvent consumption and extraction time contrast with the disadvantage of the additional cleanup of the extracts required prior to injection in the chromatographic system and the use of high-cost specialized equipment. However, due to its distinct advantages, rapid developments in MAE techniques for food BFR analysis are anticipated in the future. With regard to SFE, it is apparent that despite the demonstrated advantages, compared to PLE the high cost of the technology used and the onerous operating conditions of SFE processes has restricted its application for BFR analysis in biological samples. To the best of our knowledge, there are only three reports at the time of this publication. Wolkers’ group (2004, 2006) used SFE with the combination of HRGC-LRMS detection for the determination of PBDEs in marine mammals, and Rodil et al. (2005) investigated critical variables to optimize the extraction of PBDEs and PBBs from fish and fish feed. In addition to enhanced extraction technologies, MSPD (Martinez et al. 2005), column chromatography (Hiebl and Vetter 2007; Wang et al. 2008; Shaw et al. 2009), and classical extraction techniques such as ultrasonication (Jacobs et al. 2004) and solvent extraction (Cariou et al. 2005; Fernandes et al. 2008; Bogdal et al. 2009)

Flame Retardants

have also been tested for the determination of BFR analaysis.

Cleanup/fractionation methodologies Depending on the different matrices, a variety of sample cleanup (destructive or nondestructive) and fractionation procedures may be suitable for BFR analysis, which usually are similar to those used for the determination of other POPs (Covaci et al. 2003, 2007a). Among the cleaning techniques that attained an important position in BFR analysis, column adsorption chromatography is the most important. It is a suitable classical technique for fast, crude, extract purification after extraction with conventional techniques or as additional cleanup step for extremely coextractive loaded extracts coming from GPC treatments of animal food samples. Column chromatography was employed in most of the reviewed methods in this chapter, usually in open columns using a combination of different adsorbents. Although several types of adsorbents were available, most method development was centered on the use of acidified silica gel, Florisil, and alumina columns with different degrees of activation. Although in many applications the use of acidified silica is enough to yield sufficiently clean extracts (Roosens et al. 2008; Covaci et al. 2009b), several studies have described the use of acidified silica in combination with neutral silica (Shaw et al. 2009; Guo et al. 2007) and/or base-modified silica (Fernandes et al. 2008) in multilayer columns for improved purification. Silica gel can also be modified with alcoholic NaOH (Wolkers et al. 2006) or KOH (Berger et al. 2004), but such treatment may cause losses of Br atoms from highly brominated BFRs, such as HBCDs, PBBs, or PBDEs (Covaci et al. 2007a). BFRs are stable under strong acid conditions (de Boer et al. 2001). Thus, sulfuric acid treatment is one of the most commonly used destructive treatments in BFR analysis (Covaci et al. 2007a). Although this kind of

387

purification procedure is simple and gives satisfactory results, additional manipulation stages (several sequential LLE and centrifugation steps) in the whole process resulting in an increment of total extraction time are essential. Gel permeation chromatography (GPC) is another established method for the fractionation and/or cleanup of fatty matrices, and it is generally recommended for purifying BFR extracts obtained from complex samples. Most researchers used SX-3 BioBeads in a range of column sizes and solvents. GPC has been applied to remove lipids for PCDE analyses in fish muscle (Hajslova et al. 2007) and other marine species (van Leeuwen 2008; Mizukawa et al. 2009). A mixture of dichloromethane-cyclohexane or cyclohexane–ethyl acetate (Hajslova et al. 2007) is a typical eluent for automated GPC systems. The combination of GPC with concentrated sulfuric acid treatment (Bogdal et al. 2009) or column chromatography to remove bulk lipids was often performed (Mizukawa et al. 2009). The high degree of automation and the capability of the columns to be reused without regeneration are the key advantages of this method, particularly compared with other manual methods (e.g., column chromatography). For specific applications, isolation of the target BFR analytes from other organohalogen compounds present in the extract can be mandatory so as to minimize interference during the final determination step. There are many fractionation schemes reported in the literature, and isolation of lipid fractions generally incorporate thin-layer chromatography or column chromatography using silica gel (Martinez et al. 2005), alumina, or a combination of both (Guo et al. 2007; Fernandes et al. 2008), as well as carbon (Miyake et al. 2008) and Florisil (Ashizuka et al. 2005). Group separation or congener isolation on these materials can be achieved by using sequential elution with solvents of increasing polarity such as n-hexane and dichloromethane (Guo et al. 2007; Fernades et al. 2008). Finally,

388

Analysis of Endocrine Disrupting Compounds in Food

carbon sorbent has been used as a complementary fractionation tool to alumina (Bogdal et al. 2009).

Instrumental analysis (separation and detection) PBDEs Chromatographic-based techniques are used to identify and quantify PBDEs in foodstuffs. As can be seen in Table 17.3, gas chromatography (GC) has been widely used for determination/quantification of PBDEs by using a electron capture detector (ECD) or a mass spectrometry detector. The separation of PBDEs was carried out with a variety of capillary columns. Nonpolar GC-capillary stationary phases such as methyl polysiloxane or phenyl-methylpolysiloxane (DB-5, HP-5, ZB-5, RTX-5) were the most frequently used columns, as can be seen in Table 17.3. The use of DB-XLB (30 m × 0.25 mm I.D., 0.25 μm film thickness) and CP-Sil 13 CB (12.5 m × 0.25 mm I.D., 0.2 μm film thickness) capillary columns was also proposed for analysis of low-brominated congeners (BDE-28 to BDE-183) and for the highly brominated congeners (including BDE 203, 206, 207, 208, and 209) by GC-NCI-MS in SIM mode (Guo et al. 2007). Furthermore, AT-5 and HT-8 14% cyanopropylphenyl plus 86% dimethylpolysiloxane (BP-10) were used as stationary phases for identification and confirmation of PBDEs, methoxylated (MeO) PBDEs, and PBHDs in fish oil dietary supplements (Covaci et al. 2007b). The characteristics of the stationary phase in the GC system significantly influence the separation of the PBDE congeners. Therefore, in order to achieve enough separation between BDE congeners, in particular, and possible interferences, it is necessary to use sufficiently long columns (30–60 m) and small diameters (0.25 mm). Use of the narrow-bore capillary column (0.1 mm I.D.) also enabled good separation of all the PBDE congeners in a single gas chromatographic run in less time than was

required with conventional columns. Short columns, preferably 10–15 m, are usually used for higher BDEs, such as deca-BDE, by reducing considerably the analysis time and the exposure to high temperatures and long columns to improve the separation of lower BDEs (de Boer and Wells 2006). It is worth mentioning that despite the advances in the chromatographic systems, full congener resolution and reliable quantification of all 209 PBDEs on a single column may require significant advances in column materials and instrument programming or simply may not be possible. Splitless injection has been the most commonly used technique for GC separation because of its robustness. However, the use of splitless injection has the limitation of degradation of higher brominated BDEs at elevated temperatures, especially deca-BDE209. To overcome the above-mentioned analytical difficulties connected with the splitless mode, programmed temperature vaporizing (PTV) injectors may be used as an alternative because they allow a sample volume of up to 20−100 μL and significantly eliminate the matrix effects by releasing highboiling point coextracted compounds to the split vent and/or by trapping them in a liner. For example, the research groups of Bjorklund et al. (2003) and Tollback et al. (2003) demonstrated that PTV inlet in the cold splitless mode under optimized conditions provided sample vaporization and sample transfer into the column with excellent repeatability in comparison to a conventional splitless GC setup and without degradation of labile PBDE congeners. An alternative to PTV injection, on-column injection, may be used (Kierkegaard et al. 2004), but this technique requires very clean extracts. Analysis of PBDEs with GC-MS instruments is usually performed in electron impact (EI) mode or in electron capture negative ionization (ECNI) mode. EI mass spectra of most PBDEs have an abundant molecular ion M+ and one abundant fragment ion [M-2Br]+,

389

PBDEs (8) PBDEs (19) PBDEs (13) PBDEs (22) PBDEs (10) MeO-PBDEs & PPHDs PBDEs (8) PBDEs (4)/TBBP-A PBDEs Decabromo BDE PBDEs (15) Low-brominated congeners High-brominated congeners

PBDEs (13)/HBCDs HBCDs PBCDE PBDEs (35)/HBCD PBDEs (6)/HBCD PBDEs (11) HBCD PBDEs (10) PBDEs (16) TBBP-A/Me-TBBP-A HBCD Deca-BDE 209 PBDEs (40)

Compound

—

200

Splitless

30 ± 5 m × 0.25 ± 0.02 mm × 0.1 μm or 15 ± 1 m × 0.25 ± 0.02 mm × 0.1 μm 30 m × 0.25 mm × 0.25 μm 30 m × 0.25 mm × 0.25 μm 30 m × 0.25 mm × 0.1 μm 30 m × 0.25 mm × 0.25 μm 14 m × 0.18 mm × 0.20 μm 25 m × 0.22 mm × 0.25 μm 30 m × 0.25 mm × 0.25 μm 30 m × 25 mm × 0.25 μm 60 mm × 0.22 mm 15 m 30 m × 25 mm × 0.25 μm 12.5 m × 0.25 mm × 0.2 μm

DP-5

— —

Splitless

— —

15 m × 0.25 mm × 0.1 μm 50 m, 0.25 mm ID, 0.25 μm 15 m × 0.25 mm × 0.25 μm

DB-5 CP-Sil-8 DB-5

— — 250

250 — 280 — 250 230 280 — 260

Splitless Splitless Splitless

30 m × 0.25 mm × 0.1 μm 10 m × 0.28 mm × 0.1 μm 15 m × 0.25 mm × 0.1 μm

250 285

Source Temperature (°C)

Splitless Splitless Splitless Splitless Splitless Splitless Splitless — PTV

Splitless Splitless

15 m × 0.25 mm × 0.1 μm 30 m × 0.25 mm

DB-5 DB-5, DB-1 HP5 MS DB 5 DB-1–DB-5 DB-5

HP-5 ms RTx 5 ms DP-5ms DB-1 AT-5 HT-8 HP-5 DP-5MS DB-5MS ZB5-MS DB-XLB CP-Sil 13CB

Injection Mode

Dimensions

Column

GC–MS parameters used for the analysis of PBDEs

Table 17.3. Chromatographic parameters used for the analysis of PBDEs, HBCDs and TBBP-A.

NCI

NCI — CNI EI ECNI EI EI NCI EI

EI

EI EI ECNI EI EI ECNI

ECNI EI

Ionization

GC-MS

GC-MS Ion trap — — GC-MS GC-MS — GC-MS HRMS

HRMS

HRMS GC-MS

HRGC/HRMS HRMS GC-MS

GC-MS GC-MS

Instrument

(continued)

Guo et al. 2007

Erdogˇrul et al. 2008 Wang et al. 2008 Fernandes et al. 2008

Shelver et al. 2008 Corsolini et al. 2008 Wan et al. 2008 Cheung et al. 2008 Covaci et al. 2007b

Staskal et al. 2008

Miyake et al. 2008 van Leeuwen and de Boer 2008

Shaw et al. 2009 Bogdal et al. 2009 Roosens et al. 2008

Covaci et al. 2009b Hiebl and Vetter 2007

References

390 AcN/H2O MeOH/ H2O AcN/H2O MeOH/ H2O MeOH H2O/ MeOH AcN/H2O

MeOH/ H2O

250 mm × 4 mm, 5 μm 4.6 mm × 250 mm 150 mm × 2 mm, 5 μm 150 mm × 2.1 mm, 1.7 μm

150 mm × 2 mm, 3 μm 150 mm × 2.1 mm, 3.5 μm

100 mm × 2.1 mm, 5 μm

50 mm × 2.1 mm, 3 μm

AcN/H2O

Mobile Phase

0.25

0.15

0.15

0.2

0.5

0.2

0.5

1

0.25

Flow (mL/min)

Ammonium acetate

Ammonium acetate

Ammonium acetate Ammonium acetate

—

—

—

Acetic acid

Ammonium acetate

Mobile Phase Modifiers

ESI

ESINI

ESNI

ESNI

ESI

APCI

ESINI

APPI

ESI

Ionization

TQ

TQ

TQ

IT

TQ

TQ

TQ

QTrap

SQ

Instrument

LC-MS parameters used for the analysis of HBCDs and TBBP-A

2 mm × 150 mm, 5 μm

Dimensions

TQ, triple quadruple; SQ, single quadruple; IT, ion trap.

HPCDs TBBP-A

HBCDs TBBP-A

Hypersil C18

Varian Pursuit XRS3 Zorbax XDB-C18

HBCD (α, β, γ)

HBCD

Acquity BEH C18 Zorbax C18

Luna C18 + guard column Nucleodur 100-C8 (Interchim) C30 YMC Carotenoid S-5 Develosil C30 (Nomura)

Column

TBBP-A

TBBP-A

HBCDs

TBBP-A

HBCD TBBPA

Compound

Table 17.3. Chromatographic parameters used for the analysis of PBDEs, HBCDs and TBBP-A. (cont.)

640.6 → 79/ 640.6 → 81 540.9 → 79 540.9 → 81

300

—

—

640.6 → 79 676.7 → 640.7 555 → 543

—

—

250

—

—

150

Source Temperature (°C)

542.60 → 419.70 542.60 → 447.60 640.6

542.7 → 445.8

640.8 → 78.8

Scan

HBCD: 640.7 TBBPA: 540.9

Ion

Van Leeuwen and de Boer 2008 JohnsonRestrepo et al. 2005

Suzuki and Hasegawa 2006 Worrall et al. 2007 Roosens et al. 2008 Roosens et al. 2008

Stapleton et al. 2006

Debrauwer et al. 2005

Morris et al. 2004

Ref.

Flame Retardants

which can be used for their identification and quantification, also allowing their determination in the presence of possible coeluted compounds (such as PCBs). Despite the high selectivity of the GC-EI-MS method coming from the formation of the aforementioned fragments, it is not routinely used for PBDE analysis due to its relatively low sensitivity, especially when measuring BDE congeners with more than six bromine atoms (de Boer et al. 2000). In contrast to EI, the ECNI method is advantageous because it offers a high sensitivity for compounds with four or more bromine atoms, and it is the preferred ionization method for detection of PBDEs after separation. The sensitivity of ECNI for these compounds is approximately 10 times higher than with the use of an electron capture detector (ECD). Besides the high sensitivity, the use of LR-ECNI-MS for PBDE analysis is less selective compared to LR-EI-MS due to monitoring the bromide ions [Br]− for the homolog group; this limitation should be taken into consideration for the analysis of high-concentration samples (Covaci et al. 2008, 2009a). Several potential chromatographic interferences can hamper good quality data when working with GC–ECD or GC coupled to low-resolution mass spectrometry (GC–LRMS). The main limitation of both instrumental systems is GC coelutions among congeners of the three families (PCBs, PBDEs, and PBBs), which may cause identification problems (in ECD detection) and interference problems, increasing the background noise and LODs (in MS detection) (Gómara et al. 2006b). Special attention should be paid to the coelution between BDE 47 and CB 180 on 30-m DB-5 type columns by using ECD or GC-EI-MS (Covaci et al. 2008); therefore, identification criteria should be very restrictive (retention time and relative isotopic peak ratios). Chlorinated interferences are eliminated by NICI, but this procedure cannot recognize different brominated compounds because only bromine ions can be monitored.

391

Although bromine interferences can be resolved using a GC–MS system in electron impact (EI) ionization in a SIM mode, chlorinated or brominated compounds with equal m/z values cannot be distinguished (Eljarrat et al. 2003; Gómara et al. 2006b). Highresolution gas chromatography (HR-GC) with capillary columns is another GC technique commonly used today that allows the separation of most of the PCDE congeners. HRMS is the most selective and sensitive detection method for these kinds of analyses, but acquisition and maintenance costs are very high. Despite the high cost, HRMS is preferred over LRMS and has been used in many studies for PCDE analysis (see Table 17.3). Recently, new analytical approaches such as ITD-MS (Gómara et al. 2006c) and quadrupole ion-storage MS (QTrap-MS) (Larrazábal et al. 2004) have been evaluated for the analysis of PBDEs. These approaches may be good options because of their good analytical characteristics and the detection specificity obtained by MS/MS. A major complication of their use, however, addressed in virtually all of the relevant publications concerns the fact that coeluting halogenated congeners (e.g., PCBs) could increase the background noise and LODs, and congeners with higher bromine content could give rise to isobaric precursor and fragment ions that would contribute to the MS/MS channel monitored for a targeted PBDE. Nevertheless, both techniques may constitute low-cost, rapid, and reliable alternatives to highresolution mass spectrometry (HRMS) devices for the analysis of selected PBDEs in food samples (Covaci et al. 2007a). More and more scientists have become aware of the limitations of single-column capillary GC for the determination of PBDEs. In recent years, a new chromatographic separation approach, called comprehensive twodimensional gas chromatography (GCxGC) has been introduced as alternative to conventional GC PBDE separation because of its

392

Analysis of Endocrine Disrupting Compounds in Food

outstanding separation potential and ability to solve demanding analytical tasks. For example, Focant et al. (2004) optimized a GCxGC methodology for the determination of PCBs, PBBs, and PBDEs. Using this technique, major coelution difficulties that may arise during the simultaneous analysis of several classes of halogenated POPs could be resolved. Finally, despite the use of GC, LC-MS/MS has been recently proposed for PBDE analysis by using atmospheric pressure photoionization (APPI) as an interface (Debrauwer et al. 2005). Positive (higher sensitivity for di-BDE through penta-BDE congeners) and negative (higher sensitivity for penta-BDE through deca-BDE congeners) ion mode were both explored and found to be suitable. Although the method was not fully optimized, the first results indicated that the use of APPI opens new perspectives in terms of mass spectrometric identification of PBDEs, especially for the BDE 209 congener, given the difficulties encountered during its GC–MS analysis. HBCDs In food HBCD investigations, stereospecific differentiation combined with unequivocal identification is a vital aspect. Traditionally, HBCD has been analyzed using GC–ECNIMS, together with the PBDEs, and in much of the occurrence data HBCD is reported as total HBCD (the sum of α-, β-, and γ-HBCD diastereoisomers). However, HBCD diastereoisomers show susceptible degradation, interconversion, and adsorption during analysis at elevated temperatures (>160°C), making accurate GC-MS quantification difficult. Studies reveal that even at temperatures below 160°C, the diastereoisomers were not separated chromatographically due to peak broadening, and only total HBCD concentrations were reported (Hale et al. 2003). Consequently, due to its lack of stereoisomer specificity, the use of GC in the analysis of HBCDs should be discouraged. If GC is the

only alternative, thermal degradation of HBCDs should be minimized through cold on-column injection, short narrow-bore GC columns, thin-film stationary phases, and high carrier gas flow rates. To overcome this undesirable thermal degradation, which always took place despite the different conditions tested, and the corresponding loss of selectivity for the analysis of HBCDs, analytical methods with adequate sensitivity based on HPLC and LC-MS or LC-MS/MS should be preferentially used. Different liquid-based separation methods using LC with diode array UV-detection and electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) mass spectrometry have been proposed for the analysis of HBCDs by many authors. For example, Morris et al. (2006) developed an isomer-specific HPLC method for the simultaneous determination of the three HBCD diastereomers and the TBBP-A compound. This report illustrates quite well the advantages of the proposed HPLC method over GC-ECNI-MS, especially in terms of specificity. The major disadvantage, however, was the much lower sensitivity, which makes it less suitable for analyzing samples with extremely low HBCD concentrations. Meanwhile, Becher (2005) has shown that there are also a number of enantiomers of HBCD that can be separated and determined by HPLC using chiral columns. In addition to HPLC, reversed-phase LC coupled to ESI or APCI-MS has also been used for HBCD isomer-specific analysis in food samples. Budakowski and Tomy (2003) have compared both ESI and APCI ionization modes for the LC-MS/MS determination of the HBCD diastereomers in tissue samples. The authors concluded that LC-ESI-MS/MS is more sensitive and selective than LC– APCI-MS/MS when a single MRM for the transition [M–H]− (m/z 640.6) → [Br]− (m/z 79 and 81) is used. In contrast to the study previously described, Suzuki and Hasegawa (2006) showed that APCI ionization mode led

Flame Retardants

to a two, to five times higher signal-to-noise (S/N) ratio compared to ESI for HBCDs. The presented LC-MS/MS analytical methods combine sensitivity, specificity, speed, precision, and reliability and certainly relieve the analyst of some of the pitfalls of present GC-based methods without sacrificing performance. Despite the well-documented advantages, many of the pitfalls of LC–MS/ MS can hamper results of good quality, including coelution with other analytes or with matrix components, which can lead to ion suppression. Furthermore, the highly selective and sensitive LC-MS/MS methods in use for HBCD isomers do not permit the identification of other contaminants in the sample, even if they are more abundant than HBCD. Therefore, both GC and LC should be used to identify samples with relevant residues of technical HBCD and related BFR contaminants. TBBP-A Although TBBP-A was reported as the BFR with the highest production volume, covering around 60% of the total BFR market (BSEF 2007), it is not frequently analyzed in environmental laboratories. Its lower concentrations in biota and environmental samples and its lower bioaccumulation potential compared to PBDEs and HBCD, as well as the need for more complicated methods for a proper determination of TBBP-A, are likely to be some of the main reasons for the infrequent analysis. Essentially all of the mentioned reports show that the use of LC combined with an ultraviolet (UV) or MS detector for the determination of TBBP-A is unarguably advantageous compared to GC applications because it can be analyzed without any prior derivatization. HPLC with a UV detector has been used for the simultaneous determination of TBBP-A and octaBDE and deca-BDE (Schlummer et al. 2005) with good results and seems to be an attractive alternative to MS systems.

393

With respect to mass spectrometers that allow MS or MS/MS experiments, most research works reviewed here reported the use of quadrupole (single or triple) and iontrap instruments. Under optimized conditions, both electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces operating in negative ion (NI) ionization mode have been used for TBBP-A analysis. A major complication of the use of LC and single or tandem mass spectrometry, however, concerns the fact that separation of TBBP-A from other compounds and matrix components is greatly dependent on the mobile phase used. For example Chu et al. (2005) showed that TBBP-A proved more sensitive in methanol than in of acetonitrile, resulting in more stable detector baseline and thus in lower LOQ. Furthermore, in the case of the ESI mode, the use of mobile phase additives such as formic acid, tris(hydroxymethyl)aminomethane, and ammonium acetate for optimizing the ionization process can cause ion suppression or depression of the TBBP-A signal, depending on the additive used. In recent work, Worrall et al. (2007) reported the use of ultraperformance liquid chromatography (UPLC– ESI-MS/MS) for analysis of TBBP-A in food samples. According to the authors, the main advantage of this approach was, logically, the shorter analysis time (4 min), which can double the efficiency of the analytical method. Obviously, it is clear that the presented method provides a valuable alternative to other liquid-based separation methodologies for analyzing TBBP-A (Shi et al. 2009). The analytical methodologies used for TBBP-A determination have been recently reviewed by Covaci et al. (2009a).

Quality assurance and quality control In food analysis, assurance of high-quality data has a long history. This holds especially

394

Analysis of Endocrine Disrupting Compounds in Food

true in official food control because comparability of data is a requirement for international trade in order to avoid unnecessary court cases due to unfavorable results. The emphasis on quality assurance (QA) and quality control (QC) is therefore omnipresent and has gained considerable importance in recent years (Päpke et al. 2004). Among other things, this implies the obligation of laboratories to participate in international intercalibration exercises and to apply daily a QC program that complies with the requirements of ISO/CEI 17025 (2005) or another QA program. This means that the instruments are calibrated and their performance checked, the personnel are well trained, and all data are obtained and properly documented following written standard operating procedures (SOPs). Obviously, QA and QC procedures are of great relevance to the analysis of BFRs in a wide range of food matrices, and these procedures are being increasingly formalized and expanded worldwide. These activities are organized and promoted by many European and international programs. A good example in this area is the program Quality Assurance of Information for Marine Environmental Monitoring in Europe (QUASIMEME 2004). QUASIMEME is a proficiency testing program, which also organizes learning exercises, training programs, and workshops for “new” contaminants. This project was developed in 1993 to determine the current accuracy and improve the quality of chemical measurements made in marine monitoring programs (water, sediment, and biota matrices). The first study of QUASIMEME on BFR analysis was performed in 2001. The results showed a good agreement between the 20 participating laboratories for the BDEs 47 and 100 (2,4,6,20,40-penta BDE), whereas for other congeners, 99 (2,4,5,2040-penta BDE), 153 (2,4,5,20,40,50-hexa BDE), 154 (2,4,5,20,40,60-hexa BDE), and BDE 209, in particular, further improvement was considered necessary (de Boer and Wells 2006). Between 2001 and 2004, three further

international interlaboratory studies were organized on BFR analysis in environmental and food matrices by the Bromine Science and Environmental Forum (BSEF), Brussels, Belgium; the Netherlands Institute for Fisheries Research (RIVO), IJmuiden, The Netherlands; and QUASIMEME, with the aim of improving the quality of the analysis of BFRs. A wide range of matrices, including fish and marine mammal tissue, fish oil, shellfish, sediments, sewage sludge, human milk, and standard solutions, have been used during these exercises, enabling researchers to validate their methods and to implement reliable QC procedures. Besides PBDEs and HBCDs, TBBP-A was also included in these studies, but unfortunately very few laboratories have submitted results for TBBP-A and dimethyl TBBP-A (diMe-TBBP-A) (Covaci et al. 2009a). The results of these interlaboratory studies were summarized by de Boer and Wells (2006), who reported the main pitfalls in the analysis of BFRs. Certified reference materials (CRMs) and matrix blank materials play a vital role in verifying the accuracy of and in establishing traceability of analytical measurements. In that respect, CRMs that represent real food matrices are especially important. Various CRMs have been produced for the analysis of PBDEs in different food matrices: SRM 1588b Organics in Cod Liver Oil, SRM 1941b Organics in Marine Sediment, SRM 1944 NewYork/New Jersey Waterway Sediment, SRM 1945 Organics in Whale Blubber, SRM 1946 Lake Superior Fish Tissue, SRM 1947 Lake Michigan Fish Tissue, SRM 1974b Organics in Mussel Tissue (Mytilus edulis), SRM 2977 Mussel Tissue (Organic Contaminants and Trace Elements), and SRM 2978 Mussel Tissue (Organic ContaminantsRaritan Bay, NJ) (Stapleton et al. 2007). However, most CRMs show limitations, such as a limited number of certified contaminants, wide uncertainty ranges, concentrations (far) above the current values of interest, or a physical state not matching routine samples (e.g.,

Flame Retardants

freeze-dried materials and oils). In addition, there are no CRMs available for HBCDs and TBBP-A. Therefore, the certification of all BFR compounds in a wider range of food materials is clearly needed to address the challenges that analytical laboratories currently face (van Leeuwen et al. 2006). When CRMs are not available, the standard addition method may still be accepted as a valid approach to evaluate the accuracy and precision of the analytical procedures (Päpke et al. 2004). A QC sample spiked with at least two different (realistic) spiking levels should be used for method validation. In general terms, as far as possible, a proper incubation and aging of the spiked samples should be encouraged so that the spiked compounds simulate as closely as possible the behavior of the naturally incurred analytes (Covaci et al. 2009a). Finally, the reliability of analytical results has significantly increased with the availability of 13C-labeled standards. Additional recommendations regarding the QA/QC of BFR compounds can be found in Päpke (2004).

Flame retardants in the food chain Tables 17.4 and 17.5 summarize the concentration levels of the most important groups of BFRs in various categories of food matrices. Of the food matrices, fish appears to be the most studied food or group of species. Many studies were conducted examining the PBDE levels in fish collected from various waters, such as sea, lake, or river water. Among the data provided for all research, the congener pattern appears to give important information. BDE-47, -99, -100, -153, and -154 are generally dominant in biota worldwide and appear to have a higher potential for bioaccumulation. In most of the cases, the dominating congener is BDE-47 (ManchesterNeesvig et al. 2001; Tittlemier et al. 2004; Hajslova et al. 2007; Ashizuka et al. 2008; Roosens et al. 2008; Miyake et al. 2008; van Leeuwen and de Boer 2008; Staskal et al.

395

2008; Corsolini et al. 2008; Wan et al. 2008; Sajwan et al. 2008; Shaw et al. 2008, 2009). The levels of this tetra-BDE appear to be one order of magnitude higher than those of any other monitored congener, accounting for around 50% of the total measured PBDEs (Hajslova et al. 2007; Miyake et al. 2008; Cheung et al. 2008; Shaw et al. 2009). Whereas BDE-99 constitutes 50% of the technical penta-BDE mixture, BDE-99 contributed much less to the total in the fish, and large differences in BDE-99 abundance relative to BDE-47 were apparent between the species (Cheung et al. 2008; Roosens et al. 2008; Shaw et al. 2009). Differences in dietary exposure as well as metabolic capacity may account for the variable BDE-99 accumulation in the species. The pentaBDE-100 is relatively abundant in some species, along with the hexa-BDEs (BDE153, -154, and -155) (Tittlemier et al. 2004; Roosens et al. 2008; Sajwan et al. 2008; Shaw et al. 2008, 2009). Congener-specific differences in the uptake and biotransformation of PBDEs, taken together with the lipid content of each species, may be responsible for the observed dissimilarities. However, in some seafood samples BDE209 was the predominant congener, accounting for over 50% of all PBDEs (Miyake et al. 2008). Another study reported that BDE-209 was the predominant congener in shark samples but not in other fish samples (Johnson-Restrepo et al. 2005); however, relatively high BDE-209 concentrations have also been reported in several studies in which fish were sampled near industrial sources (Eljarrat et al. 2007). Although BDE-209 may be associated with sediment or particulate matter in the gut of some species, the presence of BDE-209 in many seafood samples is suggestive of (1) exposure to the deca-BDE mixture, (2) the lack of metabolic transformation of this congener in these seafood species, and (3) the presence of BDE-209 in sediment particles on the skin surface of the analyzed fish (Miyake et al. 2008). Many studies did

396 2003 2005 2006 2006 2006

The Netherlands, Dutch freshwaters (44)

Turkey Kahramanmaras (7)

Switzerland, Lake Thun (6)

Belgium (28)

Scheldt basin (35), Oudenaarde

2006

(8) (16) (18) (5) (42) The Netherlands (18)

2000

Spain Catalonia(2) (8) (4) (15) (3) (2) (2) 2003–2005

2006

UK (12)

(6) Spain (15)

2006

Year

EUROPE UK Scotland

Location (n)a

Fish

Oils/fats

Fish

Fruits

Fish Seafood

Seafood Oils/fats Dairy prods. Eggs Meat Oils/fats

Dairy prods. Fish Bread/cereals Meat Oils/fats Eggs Milk Vegetables Fruits Fish

Oils/fats

Seafood/ shellfish

Sample

28, 47, 49, 66, 85, 99, 100, 153, 154, 183 28, 47, 49, 66, 99, 100, 153, 154, 183

28, 47, 49, 66, 85, 99, 100, 153, 154, 183 28, 47, 49, 66, 71, 75, 77, 85, 99, 100, 119, 138, 154, 183, 190, 209 28, 47, 99, 100, 119, 153, 154, 183 28, 47, 99, 100, 153, 183

17, 28, 47, 66, 85, 99, 100, 153, 154, 183, 184, 191, 196, 197, 209

17, 28, 47, 49, 66, 71, 77, 85, 99, 100, 119, 126, 138, 153, 154, 183, 209 28, 47, 49, 66, 85, 99, 100, 153, 154, 183 Tetra, penta, hexa, hepta, and octa PDEs

Congeners

0.1–149.4 0.1–<2.3

<0.1–81 <0.1–0.8

660–11,500a

<1.3–98c

nd

0.003–0.677b 0.0148–2.958b 0.003–1.588b 0.0128–0.557b 0.007–2.518b <1.3–160c

34.1–47.9 325.3–333.9 0–35.7 102.4–109.2 569.3–587.7 58.3–64.5 13.2–16.9 5.2–7.9 0–5.8 0.024–0.880b

2270 ± 2260a

nd

— — — — — —

— — — — — — — — — —

—

<1.3–160c

Mean 0.42–0.66

ΣPBDEs

0.12–2.74

Range

nd–0.395b 0–2.482b 0–0.984b 0–0.446b 0–2.066b

0–23.4 1.4–158.3 0–17.9 12.8–24.9 30.3–169.7 0–25.8 0–8.0 0–4.0 0–2.9 nd–0.499b

—

—

Range

PBDEs

Erdogˇrul et al. 2008 Bogdal et al. 2009 Covaci et al. 2007b Roosens et al. 2008

Covaci et al. 2007b van Leeuwen and de Boer 2008

Gómara et al. 2006a

Covaci et al. 2007b Bocio et al. 2003

Fernandes et al. 2008

References

Table 17.4. Concentrations (ng/g ww/ng/g lw/ng/g fw/ng/g of oil) of PBDEs in selected food samples reported during the last decade in different countries.

397

2001–2004 2001 2002–2005 2002–2005 2002–2005 2003 2003 2003 2003–2004 2003–2004 2003–2004

USA

USA (24)

USA (18) USA (15) USA (9)

USA (9) USA (9) Dallas, Texas, (18)

Dallas, Texas, (18) Dallas, Texas, (15)

2006

USA Florida (88)

EURASIAN COUNTRIES Azerbaijan, Bulgaria, Iran, Russia

2000–2001

Fish Dairy prods.

Fish Dairy prods. Meat

Meat Dairy prods. Meat

Fish

Meat

Fish

Seafood

Fish

Fish

2005

High mountain European lakes (9)

Oils/fats

2006

Denmark, South Africa, USA, France, Sweeden (11) Mediterranean Sea (29)

Fish

Sample

2005

Year

Czech Republic Vltava and Elbe rivers (80)

Location (n)a

17, 28, 47, 66, 77, 85, 99, 100, 138, 153, 154, 183, 209

17, 28, 47, 66, 77, 85, 99, 100, 138, 153, 154, 183, 209

28/33, 47, 85, 99, 100, 153, 154, 183 17, 28, 47, 66, 77, 85, 99, 100, 138, 153, 154, 183, 209

28, 47, 66, 85, 99, 100, 138, 153, 154, 183, 203, 209

47, 99, 100, 153

28, 47, 49, 66, 85, 99, 100, 153, 154, 183 3, 5, 7, 17, 28, 47, 49, 66, 71, 77, 85, 99, 100, 119, 126, 138, 153, 154, 156 1–3, 7, 8, 10–13, 15, 17, 25, 28, 30, 32, 33, 35, 37, 47, 49, 66, 71, 75, 77, 85, 99, 100, 116, 118, 119, 126, 138, 153–155, 166, 181, 183, 190

28, 47, 49, 66, 85, 99, 100, 153, 154, 183

Congeners

nd–2.74 0.00–0.481

— — 0–0.703

— — —

—

nd–8.13a

<0.004–778a

nd–27.5

Range

PBDEs

0.011–3.726 0.008–0.683

0.009–3.078 0.001–0.679 0.039–1.426

0.039–1.426 0.008–0.683 nd–1.373

Mean

1.120 0.116

0.383

0.383 0.116

1.120

0.04 ± 0.07

618 ± 3291 2218 ± 3291

3.8 ± 8.0 7.5 ± 8.0 9.8 ± 4.7

ΣPBDEs

0.011–3.726

nd–16.33a

1.8–4190a

0.01–0.27

2.9–18.0a

<4–11206

<1.3–200c

0.9–36.0

Range

(continued)

Schecter et al. 2006

Schecter et al. 2004

JohnsonRestrepo et al. 2005 Huwe and Larsen et al. 2005 Schecter et al. 2008

Wang et al. 2008

Vives et al. 2004

Covaci et al. 2007b Corsolini et al. 2008

Hajslova et al. 2007

References

398 Seafood Fish Seafood

2003–2004 2003–2004 2003–2004 2003–2004 2005 2007 2007

CHINA Guangzhou (16)

Zhoushan (16) Guangzhou (21)

Zhoushan (21)

South China—local fishery markets (299) Bohai Bay Bohai Bay

Seafood

Fish Seafood

Fish

Fish

2003–2004

Fish Seafood

Fish

Canada, Alaska (70)

2009

Northwest Atlantic (10–20), Coast of Maine (94)

Fish

2002 2002

2006

Southern Mississippi (61, 33, 28)

Fish

Sample

CANADA Canada (49) Canada (73)

2005

Year

Savannah, Georgia (85)

Location (n)a

28, 47, 66, 85, 99, 100, 138, 153, 154, 209 28, 47, 66, 71, 99, 100, 119, 138, 153, 154, 183

47, 77, 99, 100, 105, 126, 153, 190, 209

15, 17, 28, 47, 66, 71, 75, 77, 85, 99, 100, 119, 126, 138, 153, 154, 183, 190 28, 47, 66, 85, 99, 100, 138, 153, 154

1, 2, 3, 7, 8, 10, 11, 12, 13, 15, 17, 25, 28, 30, 32, 35, 37, 47, 49, 66, 71, 75, 77, 85, 99, 100, 116, 119, 126, 138, 153, 154, 155, 156, 166, 181, 183, 190, 197, 203, 207, 209 28, 47, 49, 66, 75, 99,100, 153, 154, 155, 181, 183, 197, 203, 207, 209

28/33, 47, 66, 99, 100, 154

Congeners

nd–0.119 nd–0.029

nd–1139

0.019–12.300a

0.043–4.650a 0.038–45.6a

0.014–96.900a

0.05–42a

Range <1.0–241a

PBDEs

0.024–0.198 0.010–0.078

—

6.580–14.300a

5.020– 160.000a 1.320–10.300a 3.770–51.500a

0.42–1.37

Mean

— —

6.720a 9.200– 27.600a 3.040– 10.500a —

46.300a

62 ± 34a

3.24 ± 0.51

ΣPBDEs

0.140–5.500 0.001–5.000

18.3–81.5a

0.3–23.3

Range 10–337a

Wan et al. 2008

Guo et al. 2007

Miyake et al. 2008

Shaw et al. 2008

Tittlemier et al. 2004

Shaw et al. 2009

Sajwan et al. 2007 Staskal et al. 2008

References

Table 17.4. Concentrations (ng/g ww)/ng/g lw/ng/g fw/ng/g of oil) of PBDEs in selected food samples reported during the last decade in different countries. (cont.)

399

2001–2004 2004–2005 2004–2005 2004–2005 2005

2006

Fukuoka market Nagoya (24) Seto Island Sea (171) Kyushu (266)

Tokyo Bay

AUSTRALIA Sydney Harbour (37) Fish

Seafood

Seafood Fish Fish Fish

Fish

Sample

a Number of samples. ww, Wet weight; lw, lipid weight; fw, fat weight; nd, not detected.

2001–2004

Year

JAPAN Fukuoka market

Location (n)a

28, 47, 49, 66, 85, 99, 100, 153, 154, 183

47, 49, 66, 71, 77, 85, 99, 100, 119, 126, 138, 153, 154, 183 17, 28, 47, 49, 66, 77, 99, 100, 119, 153, 154, 183, 184, 196, 197, 206, 207, 209 3, 7, 15, 17, 28, 47, 49, 66, 71, 85, 99, 100, 153, 154, 209

Congeners

0.1–78a

nd–95.1

nd–0.106 — — —

nd–0.331

Range

PBDEs

6.4–115.4a

92.1–120a

Mean

—

—

— 0.75 0.16 0.15

—

ΣPBDEs

0.010–0.255 0.02–2.88 0.01– 0.53 0.01– 0.70

nd–1.161

Range

Losada et al. 2009

Mizukawa et al. 2009

Ashizuka et al. 2005 Ashizuka et al. 2008

References

Table 17.5. Concentrations (ng/g ww ng/g lwa) of HBCD and TBBP–A in selected food samples reported during the last decade in different countries. Location (nb)

Year

HBCD

Sample Range

EUROPE UK

2004

Fish Milk Bread/ cereal Meat Oils/fats Vegetables Fruits Dairy prods. Eggs

Range

Mean

<0.049– 0.24 <0.120– <0.280 <0.026– <0.063 <0.039– 0.150 <0.016– <0.090 <0.021– <0.610 <0.058– <0.320 <0.050– <0.120 <0.019– <0.110 0.010– 8.930 0–2000a 40–60a 390– 12100a

—

—

<0.081

—

—

<0.190

—

—

<0.043

—

—

<0.072

—

—

<0.110

—

—

<0.190

—

—

<0.054

—

—

<0.084

—

—

<0.130

0.349

—

—

— — 4500 ± 3000a

— — —

— — —

Driffield et al. 2008

2006

Seafood

Germany, Bavaria (78) Belgium, Scheldt basin (35) Oudenaarde Czech Republic, Vltava and Elbe rivers (80) The Netherlands, Dutch fresh waters North Sea (9)

2006 2006

Eggs Fish Fish

2005

Fish

nd–158

3.3 ± 3.6 27.0 ± 45.2

—

—

Hajslova et al. 2007

2003

Fish

<0.1–230

—

—

Seafood Seafood

<0.1–0.9 <30a

— —

<0.1– 1.4 — <1–35a

Fish

<0.6– 690a

—

<0.1– 245a

van Leeuwen and de Boer 2008 Morris et al. 2004

—

2009

Fish

2.4–38.1a

17.2 ± 10.2a

—

—

Shaw et al. 2009

2004– 2005 2004– 2005 2004– 2005

Fish

—

—

0.01

Ashizuka et al. 2008

Fish

—

—

Fish

—

—

nd– 0.04 nd– 0.10 nd– 0.11

2006

Seafood

<0.01– 0.20

0.12

USA Northwest Atlantic (10–20), coast of Maine (94) JAPAN Nagoya (24) Seto Island Sea (171) Kyushu (266) EURASIA Azerbaijan, Bulgaria, Iran, Russia b

Mean

References

UK, Scotland

(42)

a

TBBP–A

ng/g lipid weight. Number of samples.

400

2000– 2001 2000– 2001

—

— 11 ± 15a

Fernandes et al. 2008 Hiebl and Vetter 2007 Roosens et al. 2008

0.01 0.02

Wang et al. 2008

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not report any BDE-209 concentrations in fish (Roosens et al. 2008; van Leeuwen et al. 2008; Cheung et al. 2008). In seafood matrices other than fish, higher concentrations of PBDEs are usually observed in mussels compared to other species. Predominant congeners are BDE-47, -99, -100, and to a lesser extent -154 and -209 (Guo et al. 2007; Fernandes et al. 2008). BDE-209 usually does not correlate well with total PBDE and appears to be species selective, with the highest values occurring exclusively in mussels (Guo et al. 2007; Fernandes et al. 2008). High contribution was also observed in octopus and shrimp (Ashizuka et al. 2008). The deca-BDE formulation, which is predominately BDE-209, is still in use, and a number of studies have published occurrence data in biota for this congener (Burreau et al. 2004; de Wit et al. 2006). However, it should be noted that various pitfalls might influence the BDE-209 result. For example, the BDE-209 generally shows lower analytical recovery (40%–60%) than other BDEs (typically 60%–110%). This suggests that some removal mechanisms are active during analysis, and it is therefore essential that labeled BDE-209 is used as an internal standard in order to provide the necessary control (Fernandes et al. 2008). Moreover, due to its high molecular weight, BDE-209 has been assumed to have low bioavailability and low bioaccumulation with rapid excretion and/or biotransformation after entering the body. BDE-209 is a relatively labile substance that easily decomposes under environmental conditions yielding a large range of lower brominated PBDEs (Hajslova et al. 2007). Therefore, finding BDE-209 in various species can be attributed to sediment particles in their internal organs (Ashizuka et al. 2008). Differences in the total concentration of PBDEs among different seafood species are influenced by metabolic differences, age, and lipid content. Tittlemier et al. (2004) reported a significant positive correlation between

401

lipid content and PBDE concentrations in skinned samples of various marine fish and shellfish. Species with the highest lipid content (salmon, trout, char) also had higher concentrations of PBDEs, suggesting that lipid partitioning in the flesh drives the accumulation of PBDEs in the fish. This was in accordance with other studies (Isosaari et al. 2006; Hajslova et al. 2007; Ashizuka et al. 2008). Because PBDEs are lipophilic compounds and ubiquitous pollutants, such as PCBs, this result seems acceptable. On the other hand, other studies showed no strong PBDE-lipid correlation, suggesting that mechanisms of accumulation of PBDEs in fish muscle differ from other contaminants such as PCBs (which appear to have a strong PCB-lipid correlation), especially at low concentrations (Manchester-Neesvig et al. 2001; Shaw et al. 2008). Studies have also reported the presence of HBCD concentrations in seafood samples. Researchers usually measure total HBCD with no discrimination between the different stereoisomers of HBCD. Whereas commercial HBCD mixtures consist mainly of γHBCD (75%–89%), α-HBCD (10%–13%), and β-HBCD (1%–12%), stereoisomeric profiles of HBCDs in marine biota are dominated by α-HBCD (Morris et al. 2004; Driffield et al. 2008; van Leeuwen et al. 2008; Fernandes et al. 2008; Tomy et al. 2008), and selective enrichment of this isomer is observed with increasing trophic level in the food web (Covaci et al. 2006; Tomy et al. 2008). This may be a result of selective metabolism of the different enantiomers and/or biotransformation processes (Zegers et al. 2005). In many studies, HBCD concentrations were two- to fourfold orders of magnitude lower than PBDE concentrations in the same samples (Shaw et al. 2009). On the contrary, other studies showed higher levels of HBCDs than PBDEs, suggesting a high density of industrial activities that use HBCDs as FRs (Hajslova et al. 2007; Roosens et al. 2008; van Leeuwen et al. 2008).

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Analysis of Endocrine Disrupting Compounds in Food

TPBB-A is usually not detected above the LOD (Driffield et al. 2008; van Leeuwen et al. 2008; Fernandes et al. 2008). This low concentration can be explained by its relative low log Kow value of 4.5–5.3. Furthermore, during manufacture of TBBP-A–containing materials, the TBBP-A is chemically bonded into the polymer matrix, unlike HBCD. TBBP-A is often applied as BFR in printed circuit boards, and it is covalently bound to the epoxy resin (WHO 1995). This limits the potential for release, and leaching to the environment during the lifetime of these products is less likely. This may also account for the lack of TBBP-A in samples, although TBBP-A has been reported in sediments. No correlation was obtained between TBBP-A and fat content (Ashizuka et al. 2008). The chemical characteristics of phenolic structure and the rapid metabolic conversion of TBBP-A are probably the reasons for no correlation with fat content. Concerning other food groups, studies showed that fish exhibit the highest contamination in PBDEs, followed usually by oils, meat, and other categories such as eggs or dietary products (Bocio et al. 2003; Schecter et al. 2004, 2006; Gómara et al. 2006a; Schecter et al. 2008). Cooking can also reduce the amount of PBDEs per portion of meat or fish (Schecter et al. 2008; Perelló et al. 2009). The BDE profiles in the meat samples showed that BDE-47 and BDE-99 are the predominant congeners (Bocio et al. 2003; Schecter et al. 2004, 2006; Huwe and Larsen 2005; Gómara et al. 2006a; Schecter et al. 2008). However, BDE-209 and BDE-189, which are predominant in deca- and octa- formulations, were detected in most meat samples (more than 90%) (Gómara et al. 2006a; Schecter et al. 2006). BDE-209 appeared to be the predominant congener in some chicken samples (Gómara et al. 2006a). Higher brominated BDEs (such as BDE-183, -184, -197, or -197) were also detected in most foodstuffs, but their contribution to the total BDEs was small

(Bocio et al. 2003; Gómara et al. 2006a). The octa- and deca-BDE formulations are used in higher proportion than penta-BDE formulations in European countries, and therefore it is not surprising to find the most abundant BDEs in these formulations (BDE-183 and -209) in the analyzed samples. A possible explanation for the existence of detectable levels of BDE-184, -196, and -197 in foodstuffs, such as butter and oil samples, is that they are industrially processed food; hence, BDE 209 could be debrominated during those treatments and converted into less-brominated BDEs. In addition, these two types of samples are sometimes stored for long periods of time, and it is possible that debromination of BDE209 could take place (Gómara et al. 2006a). Studies also showed the presence of HBCD in many food matrices. In most food groups, α-HBCD was the predominant diastereoisomer. The meat-containing group saw HBCD profiles with α-HBCD predominating as opposed to the γ-diastereoisomer that dominates in the technical and commercial standards available. It has been reported that the α-HBCD predominance is a result of selective metabolism or a biotransformation process. In some food groups, no HBCD diastereoisomers were detected above the LOD. TBBP-A was also not detected above the LOD in most food matrices (Driffield et al. 2008).

Biomagnification through the food chain Studies were also conducted in order to estimate the biomagnification of the BFRs through the food chain. Measurements between prey and predator indicated the biomagnification potential of PBDEs in the marine food web (Johnson-Restrepo et al. 2005; Tomy et al. 2008; Wan et al. 2008; Shaw et al. 2009). Shaw et al. (2009) reported that concentrations of PBDEs in harbor seal blubber were two orders of magnitude higher than those in teleost fishes. Comparable

Flame Retardants

results presented by Johnson-Restrepo et al. (2005) demonstrated that concentrations of PBDE levels in sharks and dolphins were much higher than those measured in small and large fish. Therefore, lipid-normalized concentrations of PBDEs in prey fish species were compared to concentrations in their possible predators for estimation of BMFs (biomagnification factors; the ratio of concentration of contaminant between predator and prey). It should be noted that this estimate is based on the assumption that the prey fish is the sole source of the predator ’s diet. Moreover, levels of HBCDs are often elevated in species at the top of the food chain, which clearly points toward biomagnification (Morris et al. 2004; Covaci et al. 2006; Tomy et al. 2008). The BMFs of the α- and γ-HBCD stereoisomers were less than 1, suggesting a biomagnification potential of these stereoisomers. Moreover, the proportion of α-HBCD was increasing as compared to the proportion of the γ-HBCD stereoisomer in the trophic level in the food web (Covaci et al. 2006; Tomy et al. 2008). This was also in accordance with the dominance of the αHBCD stereoisomer in marine top predators (marine mammals). Variations in the solubility and partitioning behavior, as well as uptake and metabolism of individual stereoisomers, are thought to explain the enrichment of α-HBCD in aquatic organisms. Conversely, TBBP-A showed low biomagnification capacity (Morris et al. 2004). The more polar and reactive molecular properties of TBBP-A might result in a lower degree of bioaccumulation. Furthermore, in the majority of its uses, TBBP-A is chemically bound to the polymer matrix of the product into which it is applied. In these cases, potential emissions of TBBP-A from FR products to the environment are likely to be limited in comparison to other BFR compounds such as HBCD and BDEs, which are only mixed with the polymer matrix.

403

Health risks/effects There are several pathways through which humans are exposed to PBDEs. Recent research has indicated that PBDE occurrence in dust in indoor environments is an important exposure pathway, and PBDE levels in dust have been found to be positively correlated with plasma PBDE concentrations in humans (Karlsson et al. 2007). However, diet is also regarded as one of the main pathways of human exposure to PBDEs. This is confirmed in a UK study showing estimates of median PBDE exposures (sum of nine PBDEs) for UK adults of 98.7%, 0.9%, and 0.4% for food, air, and house dust, respectively (Harrad et al. 2006). The presence of PBDEs in food samples has been studied worldwide. Several studies have shown that fish and animal products provide the greatest level of dietary PBDE exposure. A clear association was found between dietary intake of fatty fish from the Baltic Sea and plasma levels of BDE-47 (Sjodin et al. 2000). A strong positive relationship between PBDE concentrations in human milk and the dietary intake of fish and shellfish was reported in Japan (Ohta et al. 2002). Anther study showed that exposure to lower brominated PBDEs and BDE-209 was unlikely to result in significant health risks to populations in the two cities in which the study was conducted (Miyake et al. 2008). In general, although a tolerable daily intake (TDI) is not set for these substances, a lowest observed adverse effect level (LOAEL) value of 1 mg/kg/day was recently suggested as reasonable for compounds or mixtures belonging to the PBDE group (Darnerud et al. 2001). Consequently, the concentrations reported in many studies did not raise any toxicological concerns (Bocio et al. 2003; Ashizuka et al. 2005; Gómara et al. 2006a; Covaci et al. 2007a; Fernandes et al. 2008; Staskal et al. 2008; Corsolini et al. 2008; Ashizuka et al. 2008).

404

Analysis of Endocrine Disrupting Compounds in Food

Scarce information is available on dietary human exposure to HBCD, although studies reporting HBCD in human blood and breast milk show that HBCD can enter the human body (Covaci et al. 2006). The UK Independent Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) has previously concluded that TDIs cannot be set for HBCD because only limited toxicological data are available (COT 2003). From a 28-day endocrine effects toxicity study with Wistar rats, a benchmark dose (based on 10% thyroid weight) of 1.6 mg/kg BW/day was derived for HBCD (van der Ven et al. 2006). Germer et al. (2006) found significant induction of drug-metabolizing enzymes in female Wistar rats in a 28-day oral exposure study at a concentration of 3.0 mg/kg BW and higher (technical HBCD mixture). In both cases, the effect level is much higher than the intake calculated in various studies (Driffield et al. 2008; van Leeuwen et al. 2008), suggesting a large margin of safety. The COT did recommend a TDI of 1 mg kg−1 BW day−1 for TBBP-A (COT 2004), and the estimated upper-bound dietary intakes of TBBP-A were well below that value estimated in various studies (Driffield et al. 2008; Ashizuka et al. 2008).

Concluding remarks and future perspectives Over the past few years, a growing number of studies have been published that have investigated the occurrence of BFRs in a range of food matrices, including fish, meat, and dairy products in Asia, North America, and Europe. Whereas in the past most studies focused on the (lower brominated) PBDEs, recent literature shows an increasing number of studies on other environmentally relevant BFRs, including TBBP-A, HBCD isomers, and higher brominated PBDEs, including the deca-congener (BDE-209). In general, the data show the presence of BFRs in food

samples and estimated that fish and animal products provide the largest amount of dietary exposure. The concentrations of BFRs are highly species specific and differ among the countries and regions of the world, probably due to different use patterns (e.g., North Americans have the highest PBDE global body burdens, averaging contaminant levels 20 times those of Europeans). The quality of the published data has much improved in the last few years and seems to be sufficient, but there are as yet no reliable data on some compounds, such as TBPA and PBDE209. Comparison of the results of occurrence studies that have been completed with different detection levels and analytical quantification (normalization or not to wet, lipid, or dry sample weight) and precision, must be done with caution if at all. Conventional techniques discussed in the previous sections, namely Soxhlet and SE, are still among the most frequently employed techniques for BFR analysis. However, for more critical analysis, the present tendency is to replace them with methodologies that are simultaneously less aggressive to the analytes and capable of dealing with them when present in ultra-low concentrations in the samples, producing more reliable data to support food safety monitoring programs. Methods should be improved and standardized and worldwide interlaboratory testing schemes should be established. Despite the advances in separation and detection of the chromatographic systems, cleanup remains important for obtaining reliable data. Some deuterated internal standards, available commercially, enhance accuracy and precision. Nevertheless, commercialization of other isotopically labeled internal standards is necessary in order to advance analytical and environmental research. Also, CRMs in a wider range of food materials is clearly needed. In the field of chromatographic analysis, important progress has been made in terms of sensitivity and selectivity. Today, GC-MS

Flame Retardants

is the most important technique for the identification and the structural characterization of target PBDE congeners, whereas a different approach may require the analysis of HBCDs and to some extent TBBP-A, for which LC-based separation techniques are often used, avoiding the derivatization step. This has the advantage of direct determination of potential metabolites of TBBP-A. As for LC-MS analysis, tandem-MS detection has largely replaced single-stage MS operation because of the much better selectivity and the wider-ranging information that can be obtained. There is still a lack of data on the levels of BFR contamination in food, animals, and human tissue to use to clarify the behavior of BFRs in metabolism and bioaccumulation and to estimate human risk in terms of these results. In particular, information about octaand deca-BDE and TBBP-A is scarce despite the importance of their surveillance. It is also clear that further work is required to obtain results with more and more precision and accuracy and at increasingly lower concentration levels of the substances. Even though the resulting concentrations of BFRs in food matrices are still lower than those of other environmental contaminants such as PCBs, recent data suggest an increasing trend in human PBDE concentrations over time. Thus, much more research efforts should be made to develop better knowledge regarding the human health risks derived from the intake of BFRs through the diet. Regarding the acute toxicity of these compounds, although according to the present knowledge toxicity seems to be relative low, studies have shown developmental and neurological effects due to PBDE exposure. The long-term effects on the balance of endocrine systems seem to present the greatest danger from these compounds. Therefore, investigations are needed to determine the potential for biological impacts on humans and aquatic organisms, including endocrine disruption.

405

References Allard A.S., Remberger M., Neilson A.H. 1987. Bacterial O-methylation of halogen-substituted phenols. Appl. Environ. Microbiol. 53:839–845. Arias P.A. 2001. Brominated flame retardants-an overview. Presented at the Second International Workshop on Brominated Flame Retardants (BFR), Stockholm, Sweden. Ashizuka Y., Nakagawa R., Tobiishi K., Hori T., Iida T. 2005. Determination of polybrominated diphenyl ethers and polybrominated dibenzo–p-dioxins/ dibenzofurans in marine products. J. Agric. Food. Chem. 53(10):3807–3813. Ashizuka Y., Nakagawa R., Hori T., Yasutake D., Tobiishi K., Sasaki K. 2008. Determination of brominated flame retardants and brominated dioxins in fish collected from three regions of Japan. Mol. Nutr. Food Res. 52:273–283. Bayen S., Lee K.H., Obbard J.P. 2004. Determination of polybrominated diphenyl ethers in marine biological tissues using microwave-assisted extraction. J. Chromatogr. A. 1035(2):291–294. Becher G. 2005. The stereochemistry of 1,2,5,6,9,10hexabromocyclododecane and its graphic representation. Chemosphere. 58:989–991. Berger U., Herzke D., Sandanger T.M. 2004. Two trace analytical methods for determination of hydroxylated PCBs and other halogenated phenolic compounds in eggs from Norwegian birds of prey. Anal. Chem. 76:441–452. Birnbaum L., Staskal D. 2004. Brominated flame retardants: Cause for concern? Environ. Health Perspect. 112(1):9–17. Bjorklund J., Tollback P., Ostman C. 2003. Large volume injection GC-MS in electron capture negative ion mode utilizing isotopic dilution for the determination of polybrominated diphenyl ethers in air. J. Sep. Sci. 26(12–13):1103–1110. Bjorklund E., Sporring S., Wiberg K., Haglund P., von Holst C. 2006. New strategies for extraction and cleanup of persistent organic pollutants from food and feed samples using selective pressurized liquid extraction. Trends Anal. Chem. 25(4):318–325. Bocio A., Llobet J.M., Domingo J.L., Corbella J., Teixido A., Casas C. 2003. Polybrominated diphenyl ethers (PBDEs) in foodstuffs: Human exposure through the diet. J. Agric. Food. Chem. 51:3191–3195. Bogdal C., Naef M., Schmid P., Kohler M., Zennegg M., Bernet D., Scheringer M., Hungerbühler K. 2009. Unexplained gonad alterations in whitefish (Coregonus spp.) from Lake Thun, Switzerland: Levels of persistent organic pollutants in different morphs. Chemosphere. 74:434–440. Bromine Science and Environmental Forum (BSEF). 2007. Available at: http://www.bsef.com. Accessed December 15, 2007. Bromine Science and Environmental Forum (BSEF). 2009. Available at: http://www.bsef.com. Accessed on February 13, 2009. Budakowski W., Tomy G. 2003. Congener-specific analysis of hexabromocyclododecane by high-

406

Analysis of Endocrine Disrupting Compounds in Food

performance liquid chromatography/electrospray tandem mass spectrometry. Rapid Comm. Mass Spectrom. 17(13):1399–1404. Burreau S., Zebuhr Y., Broman D., Ishaq R. 2004. Biomagnification of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) studied in pike (Esox lucius), perch (Perca fluviatilis) and roach (Rutilus rutilus) from the Baltic Sea. Chemosphere. 55:1043–1052. Cariou R., Antignac J-P., Marchand P., Berrebi A., Zalko D., Andre F., Le Bizec B. 2005. New multiresidue analytical method dedicated to trace level measurement of brominated flame retardants in human biological matrices. J. Chromatogr. A. 1100:144–152. Carro A.M., Lorenzo R.A., Fernández F., Phan-Tan-Luu R., Cela R. 2007. Microwave-assisted extraction followed by headspace solid-phase microextraction and gas chromatography with mass spectrometry detection (MAE-HSSPME-GC-MS/MS) for determination of polybrominated compounds in aquaculture samples. Anal. Bioanal.Chem. 388(5–6):1021–1029. Cheung K.C., Zheng J.S., Leung H.M., Wong M.H. 2008. Exposure to polybrominated diphenyl ethers associated with consumption of marine and freshwater fish in Hong Kong. Chemosphere. 70(9):1707–1720. Chu S., Haffner D., Letcher R. 2005. Simultaneous determination of tetrabromobisphenol A, tetrachlorobisphenol A, bisphenol A and other halogenated analogues in sediment and sludge by high performance liquid chromatography-electrospray tandem mass spectrometry. J. Chromatogr. A. 1097:25–32. Corsolini S., Guerranti C., Perra G., Focardi S. 2008. Polybrominated diphenyl ethers, perfluorinated compounds and chlorinated pesticides in swordfish (Xiphias gladius) from the Mediterranean Sea. Environ. Sci. Technol. 42(12):4344–4349. COT (Committee on toxicity of chemicals in food, consumer products and the environment). First draft statement on brominated flame retardants in fish from the skerne-tees river system. TOX/2003/23. COT (Committee on toxicity of chemicals in food, consumer products and the environment). Tetrabromobisphenol A—review of the toxicological data. TOX/2004/06. Cousins I., Palm A. 2003. Physical-Chemical Properties and Estimated Environmental Fate of Brominated and Iodinated Organic Compounds. Hdb Environ. Chem. 3:301–334. Covaci A., Gerecke, A.C., Law R.J., Voorspoels S., Kohler M., Heeb N.V., Leslie H., Allchin C.R., de Boer J. 2006. Hexabromocyclododecanes (HBCDs) in the environment and humans: A review. Environ. Sci. Technol. 40(12):3679–3688. Covaci A., Voorspoels S., de Boerb J. 2003. Determination of brominated flame retardants, with emphasis on polybrominated diphenyl ethers (PBDEs) in environmental and human samples—A review. Environ. Internat. 29(6):735–756. Covaci A., Voorspoels S., Ramos L., Neels H., Blust R. 2007a. Recent developments in the analysis of brominated flame retardants and brominated natural compounds. J. Chromatogr. A. 1153:145–171.

Covaci A., Voorspoels S., Vetter W., Gelbin A., Jorens P.G., Blust R., Neels H. 2007b. Anthropogenic and naturally occurring organobrominated compounds in fish oil dietary supplements. Environ. Sci. Technol. 41(15):5237–5244. Covaci A., Voorspoels S., D’Silva K., Huwe J., Harrad S. 2008. Brominated flame retardants as food contaminants. Comp. Anal. Chem. 51:507–570. Covaci A., Voorspoels S., Mohamed Abou-Elwafa A., Geens T., Harrad S., Law R.J. 2009a. Analytical and environmental aspects of the flame retardants tetrabromobisphenol-A and its derivatives. J. Chromatogr. A. 1216:346–363. Covaci A., Roosensa L., Dirtua A.C., Waegeneersd N., van Overmeiree I., Neelsa H., Goeyense L. 2009b. Brominated flame retardants in Belgian homeproduced eggs: Levels and contamination sources. Sci. Total Environ. 407:4387–4396. Darnerud P.O., Eriksen G.S., Johannesson T., Larsen P.B., Viluksela M. 2001. Polybrominated diphenyl ethers: Occurrence, dietary exposure, and toxicology. Environ. Health Perspect. 109(Suppl1):49–68. Darnerud P.O. 2003. Toxic effects of brominated flame retardants in man and in wildlife. Environ. Int. 29:841–853. Davis J.W., Gonsior S.J., Marty G., Ariano J.M. 2005. The transformation of hexabromocyclododecane in aerobic and anaerobic soils and aquatic sediments. Water Res. 39:1075–1084. Davis J.W., Gonsior S.J., Markham D.A., Friederich U., Hunziker R.W., Ariano J.M. 2006. Biodegradation and product identification of [14c]hexabromocyclododecane in wastewater sludge and freshwater aquatic sediment. Environ. Sci. Technol. 40:5395– 5401. de Boer J., de Boer K., Boon J.P. 2000. Polybrominated biphenyls and diphenylethers. The Handbook of Environmental Chemistry, vol. 3, Part K: New Types of Persistent Halogenated Compounds. Berlin: Springer, pp. 62–95. de Boer J., Allchin C., Law R., Zegers B., Boon J.P. 2001. Method for the analysis of polybrominated diphenyl ethers in sediments in biota. Trends Anal. Chem. 20:591–599. de Boer, J., Wells D.E. 2006. Pitfalls in the analysis of brominated flame retardants in environmental, human and food samples–including results of three international interlaboratory studies. Trends Anal. Chem. 25(4):364–372. Debrauwer L., Riu A., Jouahri M., Rathahao E., Jouanin I., Antigna J-P., Cariou R., Le Bize B., Zalko D. 2005. Probing new approaches using atmospheric pressure photo ionization for the analysis of brominated flame retardants and their related degradation products by liquid chromatography-mass spectrometry. J. Chromatogr. A. 1082(1 Spec. Iss.):98–109. de Wit C.A. 2002. An overview of brominated flame retardants in the environment. Chemosphere. 46:583–624. de Wit C.A., Alaee M., Muir D.C.G. 2006. Levels and trends of brominated flame retardants in the Arctic. Chemosphere. 64:209–233.

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Directive 2003/11/EC of the European Parliament and of the Council of 6 February 2003. Official Journal of the European Union, 15.2.2003, L 42/45, 2003. Directive 2002/96/EC of the European Parliament and of the Council of 27 January 2003 on waste electrical and electronic equipment (WEEE), Official J. European Union L37 (2003) 24–38. Driffield M., Harmer N., Bredley E., Fernandes A.R., Rose M., Mortimer D., Dicks P. 2008. Determination of brominated flame retardants in food by LC-MS/MS: Diastereoisomer-specific hexabromocyclododecane and tetrabromobisphenol A. Food Addit. Contam. 25:895–903. Eljarrat E., de la Cal A., Barceló D. 2003. Potential chlorinated and brominated interferences on the polybrominated diphenyl ether determinations by gas chromatography–mass spectrometry. J. Chromatogr. A. 1008:181–192. Eljarrat E., Labandeira A., Marsh G., Raldïa D., Barcelo D. 2007. Decabrominated diphenyl ether in river fish and sediment samples collected downstream an industrial park. Chemosphere. 69:1278–1286. Eljarrat E., Barceló D. 2009. Chlorinated and brominated organic pollutants in contaminated river sediments. Hdb Environ. Chem. (5): Water Pollut. 5 T: 21–56. Erdogˇrul Ö. 2008. Pesticide residues in liquid pekmez (grape molasses). Environ. Monit. Assess. 144:323– 328. Eriksson P., Viberg H., Fischer C., Wallin M., Fredriksson A. 2002. A comparison on developmental neurotoxic effects of hexabromocyclododecane, 2,2′4,4′,5,5′hexabromodiphenylether (PBDE 153) and 2,2′,4,4′,5,5′ hexachlorobiphenyl (PCB 153). Organohalogen Compounds 57:389–392. European Commission (EC). 2001. Council Regulation (EC) 2375/2001, Amending Commission Regulation (EC) 466/2001. Off. J. Eur. Communities 2001, L 321/1–5. European Flame Retardant Association (EFRA). Available at: http://www.flameretardants.eu/Content/ Default.asp? Accessed on June 10, 2010. European Flame Retardant Association (EFRA). 2006. Advise of the scientific panel on contaminants in the food chain on a request from the commission related to relevant chemical compounds in the group of brominated flame retardants for monitoring feed and food (Question N8 EFSAQ- 2005-244). EFSA J. 328:1–4. European Union (EU). Court proceedings 2008/C 116/04 of the Court of Justice of the European Communities. Off. J. Eur. Union C116/2-3, 2008. Existing Substances Regulation. 1993. ESR, 93/793/ EEC. Council Regulation (EEC) No. 793/93 of 23 March 1993 on the evalution and control of the risks of existing substances, Official Journal of the European Communities, L/84(36):1–75. Fajar N.M., Carro A.M., Lorenzo R.A., Fernandez F., Cela R. 2008. Optimization of microwave-assisted extraction with saponification (MAES) for the determination of polybrominated flame retardants in aquaculture samples. Food Addit. Contam. 25(8):1015– 1023.

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Fernandes A., Dicks P., Mortimer D., Gem M., Smith F., Driffield M., White S., Rose M. 2008. Brominated and chlorinated dioxins, PCBs and brominated flame retardants in Scottish shellfish: Methodology, occurrence and human dietary exposure. Mol. Nutr. Food Res. 52:238–249. Focant J-F., Sjodin A., Turner W.E., Patterson D.G. 2004. Measurement of selected polybrominated diphenyl ethers, polybrominated and polychlorinated biphenyls, and organochlorine pesticides in human serum and milk using comprehensive two-dimensional gas chromatography isotope dilution time-of-flight mass spectrometry. Anal. Chem. 76:6313–6320. Germer S., Piersma A.H., van der Ven L., Kamyschnikow A., Fery Y., Schmitz H-J., Schrenk D. 2006. Subacute effects of the brominated flame retardants hexabromocyclododecane and tetrabromobisphenol-A on hepatic cytochrome P450 levels in rats. Toxicology. 218:229– 236. Gómara B., Herreo L., Gonzalez M.J. 2006a. Survey of polybrominated diphenyl ether levels in Spanish commercial foodstuffs. Environ. Sci. Technol. 40:7541–7547. Gómara B., Garcıa-Ruiz C., Gonzalez Marıa J., Marina M.L. 2006b. Fractionation of chlorinated and brominated persistent organic pollutants in several food samples by pyrenyl-silica liquid chromatography prior to GC–MS determination. Anal. Chim. Acta. 565:208–213. Gómara B., Herrero L., Bordajandi L., González M.J. 2006c. Quantitative analysis of polybrominated diphenyl ethers in adipose tissue, human serum and foodstuff samples by gas chromatography with ion trap tandem mass spectrometry and isotope dilution. Rapid Commun. Mass Spectrom. 20(2):69–74. Guo J-Y., Wu F-C., Mai B-X., Luo X-J., Zeng E.Y. 2007. Polybrominated diphenyl ethers in seafood products of South China. J. Agric. Food. Chem. 55(22): 9152–9158. Hajslova J., Pulkrabova J., Poustka J., Cajka T., Randak T. 2007. Brominated flame retardants and related chlorinated persistent organic pollutants in fish from river Elbe and its main tributary Vltava. Chemosphere. 69:1195–1203. Hakk H., Letcher R.J. 2003. Metabolism in the toxicokinetics and fate of brominated flame retardants–a review. Environ. Int. 29:801–828. Hale R., Alaee M., Manchester-Neesvig J., Stapletond H., Ikonomou M. 2003. Polybrominated diphenyl ether flame retardants in the North American environment. Environ. Int. 29(6):771–779. Hale R.C., La Guardia M.J., Harvey E., Gaylor M.O., Mainor T.M. 2006. Brominated flame retardant concentrations and trends in abiotic media. Chemosphere. 64(2):181–186. Hamers T., Kamstra J.H., Sonneveld E., Murk A.J., Kester M.H.A., Andersson P.L., Legler J., Brouwer A. 2006. In vitro profiling of the endocrine disrupting potency of brominated flame retardants. Toxicol. Sci. 92:157–173. Harrad S., Hazrati S., Ibarra C. 2006. Concentrations of polychlorinated biphenyls in indoor air and

408

Analysis of Endocrine Disrupting Compounds in Food

polybrominated diphenyl ethers in indoor air and dust in Birmingham, United Kingdom: Implications for human exposure. Environ. Sci. Technol. 40:4633– 4638. Helleday T., Tuominen K.-L., Bergman Å., Jenssen D. 1999. Brominated flame retardants induce intragenic recombination in mammalian cells. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 439 (2):137–147. Hiebl J., Vetter W. 2007. Detection of hexabromocyclododecane and its metabolite pentabromocyclododecene in chicken egg and fish from the official food control. J. Agric. Food. Chem. 55(9):3319–3324. Hites R.A. 2004. Polybrominated diphenyl ethers in the environment and in people: A meta-analysis of concentrations. Environ. Sci. Technol. 38:945–956. Hites R.A. 2006. Brominated flame retardants in the Great Lakes. Handbook of Environmental Chemistry Vol. 5. Part N. Springer-Verlag: Berlin & Heidelberg, Germany, pp. 355–390. Huwe J.K., Larsen G.L. 2005. Polychlorinated dioxins, furans, and biphenyls, and polybrominated diphenyl ethers in a U.S. meat market basket and estimates of dietary intake. Environ. Sci. Technol. 39 (15):5606– 5611. International Organization for Standardization (ISO/ IEC). 17025. 2005. General Requirements for the Competence of Testing and Calibration Laboratories, 2nd ed. International Organization for Standardization (ISO), Geneva, Switzerland. Isosaari P., Hallikainen A., Kiviranta H., Vuorinen P.J., Parmanne R., Koistinen J., Vartiainen T. 2006. Polychlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls, naphthalenes and polybrominated diphenyl ethers in the edible fish caught from the Baltic Sea and lakes in Finland. Environ. Pollut. 141:213–225. Jacobs M.N., Covaci A., Gheorghe A., Schepens P. 2004. Time trend investigation of PCBs, PBDEs, and organochlorine pesticides in selected n-3 polyunsaturated fatty acid rich dietary fish oil and vegetable oil supplements: Nutritional relevance for human essential n-3 fatty acid requirements. J. Agric. Food Chem. 52:1780–1788. Janssen S. 2005. Brominated flame retardants rising levels of concern. Health Care With Out Harm (HCWH): Arlington, VA, USA. Johnson-Restrepo B., Kannan K., Addink R., Adams D.H. 2005. Polybrominated diphenyl ethers and polychlorinated biphenyls in a marine foodweb of coastal Florida. Environ. Sci. Technol. 39:8243–8250. Karlsson M., Julander A., van Bavel B., Hardell L. 2007. Levels of brominated flame retardants in blood in relation to levels in household air and dust. Environ. Int. 33:62–69. Kester M.H., Bulduk S., van Toor H., Tibboel D., Meinl W., Glatt H., Falany C.N., Coughtrie M.W., Schuur A.G., Brouwer A., Visser T.J. 2002. Potent inhibition of estrogen sulfotransferase by hydroxylated metabolites of polyhalogenated aromatic hydrocarbons reveals alternative mechanism for estrogenic activity of endocrine disrupters. J. Clin. Endocrinol. Metab. 87:1142–1150.

Kierkegaard, A., Bjorklund J., Friden U. 2004. Identification of the flame retardant decabromodiphenyl ethane in the environment. Environ. Sci. Technol. 38:3247–3253. Kitamura S., Jinno N., Ohta S., Kuroki H., Fujimoto N. 2002. Thyroid hormonal activity of the flame retardants tetrabromobisphenol A and tetrachlorobisphenol A. Biochem. Biophys. Res. Commun. 293:554– 559. Larrazábal D., Martınez M.A., Eljarrat E., Barcelo D., Fabrellas B. 2004. Optimization of quadrupole ion storage mass spectrometric conditions for the analysis of selected polybrominated diphenyl ethers. Comparative approach with negative chemical ionization and electron impact mass spectrometry. J. Mass Spectrom. 39:1168–1175. Law R.J., Allchin C.R., de Boer J., Covaci A., Herzke D., Lepom P., Morris S., Tronczynski J., de Wit C.A. 2006. Levels and trends of brominated flame retardants in the European environment. Chemosphere. 64:187–208. Law R.J., Herzke D., Harrad S., Morris S., Bersuder P., Allchin C.R. 2008. Levels and trends of HBCD and BDEs in the European and Asian environments, with some information for other BFRs. Chemosphere. 73(2):223–241. Legler J. 2008. New insights into the endocrine disrupting effects of brominated flame retardants. Chemosphere. 73(2):216–222. Li Y., Wang T., Hashi Y., Haifang L., Lin J-M. 2009. Determination of brominated flame retardants in electrical and electronic equipments with microwaveassisted extraction and gas chromatography-mass spectrometry. Talanta. 78(4–5):1429–1435. Losada S., Roach A., Roosens L., Santos F.J., Galceran M.T., Vetter W., Neels H., Covaci A. 2009. Biomagnification of anthropogenic and naturallyproduced organobrominated compounds in a marine food web from Sydney Harbour, Australia. Environ. Int. 35(8):1142–1149. Manchester-Neesvig J.B., Valters K., Sonzogni W.C. 2001. Comparison of polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) in Lake Michigan salmonids. Environ. Sci. Technol. 35(6):1072–1077. Mariussen E., Fonnum F. 2003. The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles. Neurochem. Int. 43:533–542. Mariussen E., Fonnum F. 2006. Neurochemical targets and behavioral effects of organohalogen compounds: An update. Crit. Rev. Toxicol. 36(3):253–289. Martínez A., Ramil M., Montes R., Hernanz D., Rubí E., Rodríguez I., Cela R. 2005. Development of a matrix solid-phase dispersion method for the screening of polybrominated diphenyl ethers and polychlorinated biphenyls in biota samples using gas chromatography with electron-capture detection. J. Chromatogr. A. 1072:83–91. Meerts I., Letcher R.J., Hoving S., Marsh G., Bergman A., Lemmen J.G., van der Burg B., Brouwer A. 2001. In vitro estrogenicity of polybrominated diphenyl

Flame Retardants

ethers, hydroxylated PBDEs, and polybrominated bisphenol A compounds. Environ. Health Perspect. 10:399–407. Miyake Y., Jiang Q., Yuan W., Hanari N., Okazawa T., Wyrzykowska B., So Man K., Lam Paul K.S., Yamashita N. 2008. Preliminary health risk assessment for polybrominated diphenyl ethers and polybrominated dibenzo-p-dioxins/furans in seafood from Guangzhou and Zhoushan, China. Mar. Pollut. Bull. 57:357–364. Mizukawa K., Takada H., Takeuchi I., Ikemoto T., Omori K., Tsuchiya K. 2009. Bioconcentration and biomagnification of polybrominated diphenyl ethers (PBDEs) through lower-trophic-level coastal marine food web. Mar. Pollut. Bull. 58(8):1217–1224. Morris S., Allchin C.R., Zegers B.N., Haftka J.J.H., Boon J.P., Belpaire C., Leonards P.E.G., van Leeuwen S.P.J., de Boer J. 2004. Distribution and fate of HBCD and TBBPA brominated flame retardants in North Sea estuaries and aquatic food webs. Environ. Sci. Technol. 38(21):5497–5504. Morris S., Bersuder P., Allchin C.R., Zegers B., Boon J.P., Leonards P.E.G., de Boer J. 2006. Determination of the brominated flame retardant, hexabromocyclodocane, in sediments and biota by liquid chromatography-electrospray ionisation mass spectrometry. Trends Analyt. Chem. 25:343–349. MSFR (Market Study Flame Retardants). Ceresana Research: Konstanz Germany, 2008. Available at: www.ceresana.com/en/market - studies/additives/ flame-retardants Accessed on February 4, 2009. Ohta S., Ishizuka D., Nishimura H., Nakao T., Aozasa O., Shimidzu Y., Ochiai F., Kida T., Nishi M., Miyata H. 2002. Comparison of polybrominated diphenyl ethers in fish, vegetables, and meats and levels in human milk of nursing women in Japan. Chemosphere. 46:689–696. Päpke O., Furst P., Herrmann T. 2004. Determination of polybrominated diphenylethers (PBDEs) in bioogical tissues with special emphasis on QC/QA measures. Talanta. 63:1203–1211. Perelló G., Martí-Cid R., Castell V., Llobet J.M., Domingo J.L. 2009. “Concentrations of polybrominated diphenyl ethers, hexachlorobenzene and polycyclic aromatic hydrocarbons in various foodstuffs before and after cooking.” Food and Chemical Toxicology 47(4):709–715. QUASIMEME (Quality Assurance of Information for Marine Environmental Monitoring in Europe) 2004. Available at: www.quasimeme.marlab.ac.uk/. Rayne S. 2009. Comment on “Polybrominated diphenyl ether flame retardants in the North American environment.” Environ. Int. 35(2):450. Rayne S., Ikonomou M.G., Whale M.D. 2003. Anaerobic microbial and photochemical degradation of 4,4′-dibromodiphenyl ether. Water Res. 37:551– 560. Regueiro J., Llompart M., Garcia-Jares C., Cela R. 2006. Determination of polybrominated diphenyl ethers in domestic dust by microwave-assisted solvent extraction and gas chromatography-tandem mass spectrometry. J. Chromatogr. A. 1137(1):1–7.

409

Regueiro, J., Llompart M., Garcia-Jares C., Cela R. 2007. Factorial-design optimization of gas chromatographic analysis of tetrabrominated to decabrominated diphenyl ethers. Application to domestic dust. Anal. Bioanal. Chem. 388(5–6):1095–1107. Rodil R., Carro A.M., Lorenzo R.A., Torrijos R. 2005. Selective extraction of trace levels of polychlorinated and polybrominated contaminants by supercritical fluid-solid-phase microextraction and determination by gas chromatography/mass spectrometry. Application to aquaculture fish feed and cultured marine species. Anal. Chem. 77:2259–2265. Ronen Z., Abeliovich A. 2000. Anaerobic-aerobic process for microbial degradation of tetrabromobisphenol A. Appl. Environ. Microbiol. 66:2372–2377. Roosens L., Dirtu A.C., Goemans G., Belpaire C., Gheorghe A., Neels H., Blust R., Covaci A. 2008. Brominated flame retardants and polychlorinated biphenyls in fish from the river Scheldt, Belgium. Environ. Int. 34:976–983. Sajwan K.S., Kumar K.S., Nune S., Fowler A., Richardson J.P., Loganathan B.G. 2008. Persistent organochlorine pesticides, polychlorinated biphenyls, polybrominated diphenyl ethers in fish from coastal waters off Savannah, GA, USA. Toxicol. Environ. Chem. 90(1):81–96. Schecter A., Harris T.R., Shah N., Musumba A., Päpke O. 2008. Brominated flame retardants in US food. Mol. Nutr. Food Res. 52:266–272. Schecter A., Päpke O., Harris T.R., Tung K-C., Musumba A., Olson J., Birnbaum L. 2006. Polybrominated diphenyl ether (PBDE) levels in an expanded market basket survey of U.S. Food and estimated PBDE dietary intake by age and sex. Environ. Health Perspect. 114:1515–1520. Schecter A., Päpke O., Tung K-C., Staska D., Birnbaum L. 2004. Polybrominated diphenyl ethers contamination of United States food. Environ. Sci. Technol. 38:5306–5311. Schecter A., Vuk M.P., Päpke O., Ryan J.J., Birnbaum L., Rosen R. 2003. Polybrominated diphenyl ethers (PBDEs) in US mothers’ milk. Environ. Health Perspect. 111:1723–1729. Schlummer M., Brandl F., Mäurer A., Eldik R. 2005. Analysis of flame retardant additives in polymer fractions of waste of electric and electronic equipment (WEEE) by means of HPLC–UV/MS and GPC– HPLC–UV. J. Chromatogr. A. 1064(1):39–51. Shaw, S.D., Berger M.L., Brenner D., Carpenter D.O., Tao L., Hong C-S., Kannan K. 2008. Polybrominated diphenyl ethers (PBDEs) in farmed and wild salmon marketed in the Northeastern United States. Chemosphere. 71:1422–1431. Shaw S.D., Berger M.L., Brenner D., Kannan K., Lohmann N., Päpke O. 2009. Bioaccumulation of polybrominated diphenyl ethers and hexabromocyclododecane in the northwest Atlantic marine food web. Sci. Total Environ. 407:3323–3329. Shelver W.L., Parrotta C.D., Slawecki R., Li Q.X., Ikonomou M.G., Barcelo D., Lacorte S., Rubio F.M. 2008. Development of a magnetic particle immunoassay for polybrominated diphenyl ethers and

410

Analysis of Endocrine Disrupting Compounds in Food

application to environmental and food matrices. Chemosphere. 73:S18–S23. Shi Z.-X., Wu Y.-N., Li J.-G., Zhao Y.-F., Feng J.-F.G. 2009. Dietary exposure assessment of Chinese adults and nursing infants to tetrabromobisphenol-A and hexabromocyclododecanes: Occurrence measurements in foods and human milk. Environ. Sci. Techn. 43(12):4314–4319. Sjodin A., Hagmar L., Klasson-Wehler E., Bjork J., Bergman E. 2000. Influence of the consumption of fatty Baltic Sea fish on plasma levels of halogenated environmental contaminants in Latvian and Swedish men. Environ. Health Perspect. 108:1035–1041. Stapleton H.M., Dodder N.G., Kucklick J.R., Reddy C.M., Schantz M.M., Becker P.R., Gulland, F., Porter B.J., Wise S.A. 2006. Determination of HBCD, PBDEs and MeO-BDEs in California sea lions (Zalophus californianus) stranded between 1993 and 2003. Mar. Pollut. Bull. 52(5):522–531. Stapleton H.M., Keller J.M., Schantz M.M., Kucklick J.R., Leigh S.D., Wise S.A. 2007. Determination of polybrominated diphenyl ethers in environmental standard reference materials. Anal. Bioanal Chem. 387:2365–2379. Staskal D.F., Scott L.L.F., Haws L.C., Luksemburg W.J., Birnbaum L.S., Urban J.D., Williams E.S., Paustenbach D.J., Harris M.A. 2008. Assessment of polybrominated diphenyl ether exposures and health risks associated with consumption of Southern Mississippi catfish. Environ. Sci. Technol. 42(17):6755–6761. Strack S., Detzel T., Wahl M., Kuch B., Krug H.F. 2007. Cytotoxicity of TBBPA and effects on proliferation, cell cycle and MAPK pathways in mammalian cells. Chemosphere. 67:S405–S411. Suzuki, S., Hasegawa A. 2006. Determination of hexabromocyclododecane diastereoisomers and tetrabromobisphenol A in water and sediment by liquid chromatography/mass spectrometry. Anal. Sci. 22(3):469–474. Talsness C.E. 2008. Overview of toxicological aspects of polybrominated diphenyl ethers: A flame-retardant additive in several consumer products. Environ. Res. 108(2):158–167. Tittlemier S.A., Forsyth D., Breakell K., Verigin V., Ryan J.J., Hayward S. 2004. Polybrominated diphenyl ethers in retail fish and shellfish samples purchased from Canadian markets. J. Agric. Food Chem. 52:7740–7745. Tollback P., Bjorklund J., Ostman C. 2003. Large-volume programmed-temperature vaporiser injection for fast gas chromatography with electron capture and mass spectrometric detection of polybrominated diphenyl ethers. J. Chromatogr. A. 991(2):241– 253. Tomy G.T., Pleskach K., Oswald T., Halldorson T., Helm P.A., MacInnis G., Marvin C.H. 2008. Enantioselective bioaccumulation of hexabromocyclododecane and congener-specific accumulation of brominated diphenyl ethers in an eastern Canadian Arctic marine food web. Environ. Sci. Technol. 42(10):3634–3639.

van der Ven L.T.M., Verhoef A., Kuil T., Slob W. 2006. A 28-day oral dose toxicity study enhanced to detect endocrine effects of hexabromocyclododecane in Wistar rats. Toxicol. Sci. 94:281–292. van Leeuwen S.P.J., van Cleuvenbergen R., Abalos M., Pasini A-L., Eriksson U., Cleemann M., Hajslova J., de Boer J. 2006. New certified and candidate certified reference materials for the analysis of PCBs, PCDD/ Fs, OCPs and BFRs in the environment and food. Trends Anal. Chem. 25(4):397–409. van Leeuwen S.P.J., de Boer J. 2008. Brominated flame retardants in fish and shellfish–levels and contribution of fish consumption to dietary exposure of Dutch citizens to HBCD. Mol. Nutr. Food Res. 52: 194–203. Vilaplana F., Karlsson P., Ribes-Greus A., Ivarsson P., Karlsson S. 2008. Analysis of brominated flame retardants in styrenic polymers. Comparison of the extraction efficiency of ultrasonication, microwaveassisted extraction and pressurised liquid extraction. J. Chromatogr. A. 1196–1197(1–2):139–146. Vilaplana F., Ribes-Greus A., Karlsson S. 2009. Microwave-assisted extraction for qualitative and quantitative determination of brominated flame retardants in styrenic plastic fractions from waste electrical and electronic equipment (WEEE). Talanta. 78(1): 33–39. Vives I., Grimalt J.O., Lacorte S., Guillamon M., Barcelo D., Rosseland B.O. 2004. Polybromodiphenyl ether flame retardants in fish from lakes in European high mountains and Greenland. Environ. Sci. Technol. 38:2338–2344. Voorspoels S., Covaci A., Neels H., Schepens P. 2007. Dietary PBDE intake: A market-basket study in Belgium. Environ. Int. 33:93–97. Wan Y., Hu J., Zhang K., An L. 2008. Trophodynamics of olybrominated diphenyl ethers in the marine food web of Bohai Bay, North China. Environ. Sci. Technol. 42(4):1078–1083. Wang W., Batterman S., Chernyaka S., Nriagua J. 2008. Concentrations and risks of organic and metal contaminants in Eurasian caviar. Ecotoxicol. Environ. Saf. 71(1):138–148. Ward J., Mohapatra S.P., Mitchell A. 2008. An overview of policies for managing polybrominated diphenyl ethers (PBDEs) in the Great Lakes basin. Environ. Int. 34:1148–1156. WHO (World Health Organization). Tetrabromobisphenol A and derivatives. Environmental Health Criteria 172, 1995. Wolkers H., van Bavel B., Derocher A.E., Wiig Ø., Kovacs K.M., Lydersen C., Lindstrom G. 2004. Congener-specific accumulation and food chain transfer of polybrominated diphenyl ethers in two Arctic food chains. Environ. Sci. Technol. 38:1667– 1674. Wolkers H., Hammill M.O., van Bavel B. 2006. Tissuespecific accumulation and lactational transfer of polychlorinated biphenyls, chlorinated pesticides, and brominated flame retardants in hooded seals (Cistophora cristata) from the Gulf of St.

Flame Retardants

Lawrence: Applications for monitoring. Environ. Pollut. 142:476–486. Worrall K., Hancock P., Fernandes A., Driffield M. 2007. Enhanced separation and detection of tetrabromobisphenol-a and hexabromocyclododecane isomers using UPLC/MS/MS. Organohalogen Comp 67:698–701. Yamada-Okabe T., Sakai H., Kashima Y., YamadaOkabe H. 2005. Modulation at a cellular level of the thyroid hormone receptor-mediated gene expression by 1,2,5,6,9,10-hexabromocyclododecane (HBCD),

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4,4′-diiodobiphenyl (DIB), and nitrofen (NIP). Toxicol. Lett. 155(1):127–133. Yogui G.T., Sericano J.L. 2009. Polybrominated diphenyl ether flame retardants in the U.S. marine environment: A review. Environ. Int. 35(3):655–666. Zegers B.N., Mets A., van Bommel R., Minkenberg C., Hamers T., Kamstra J.H., Pierce G.J., Boon J.P. 2005. Levels of HBCD in harbour porpoises and common dolphin from western European seas with evidence for stereospecific biotransformation by cytochrome. Environ. Sci. Technol. 39:2095–2100.

Chapter 18 Personal Care Products Guang-Guo Ying

Introduction Personal care products are chemicals that have direct use by consumers on the human body (not intended for ingestion, with the exception of food supplements) and are generally directed at altering odor, appearance, touch, or taste, yet do not display significant biochemical activity (Daughton and Ternes 1999). These chemicals are most commonly used as the active ingredients or preservatives in cosmetics, toiletries, or fragrances, whereas some may be intended to prevent diseases, for example sunscreen agents. The use of personal care products is often widespread; for example, in Germany the combined annual output for eight categories of personal care products has been estimated at 559,000 tons in 1993 (Daughton and Ternes 1999). Due to their widespread use, some personal care products, such as disinfectants/antiseptics, musk fragrances, preservatives/antioxidants, and sunscreen agents, have been reported in the environment and in food as well (e.g., Balmer et al. 2005; Kolpin et al. 2002; Wang et al. 2006; Ying and Kookana 2007). Some of these personal care products have been demonstrated to have potential toxic or endocrine-disrupting effects on organisms (Alslev et al. 2005; Chen et al. 2008; Darbre 2006; Schlumpf et al. 2008a; Yang et al. 2008).

Analysis of personal care products in food can provide human exposure data for risk assessment. This chapter will discuss analytical techniques for the following categories of personal care products (Table 18.1): 1. 2. 3.

4.

Disinfectants/antiseptics: triclosan and triclocarban; Musk fragrances: polycyclic musks, nitro musks, and their metabolites; Preservatives/antioxidants: preservatives such as sorbic acid, benzoic acid, and p-hydroxybenzoic acid esters (parabens), as well as antioxidants such as tertbutyl-4-hydroxyanisole (BHA), 2,6di-tert-butyl-p-hydroxytoluene (BHT), α-tocopherol (α-T), α-tocopheryl acetate (α-TA), tert-butylhydroquinone (TBHQ), 2,4,5-trihydroxybutylrophenone (THBP); Sunscreen agents: ultraviolet (UV)absorbing compounds (UV filters) and their metabolites, including benzophenone-3 (BP3), octyl-dimethyl-p-aminobenzoic acid (OD-PABA), homosalate (HMS), 2-octyl salicylate (EHS), octyl methoxycinnamate (OMC), isoamyl p-methoxycinnamate (IMC), butyl methoxydibenzoyl methane (BMDBM), 4-methylbenzlidene camphor (4-MBC), and octocrylene (OC).

Sample preparation Disinfectants/antiseptics Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

The disinfectants and antiseptics analyzed in foodstuffs are mainly two compounds: 413

Table 18.1. Structure and physicochemical parameters of common personal care products. Trade Name and CAS Name and Number

Structure

Disinfectants Triclosan 2,4,4′-trichloro-2′hydroxydiphenyl ether (TCS) CAS 3380-34-5

Cl

OH

Log Kow

289.5

4.7

4.621

315.6

4.9

0.6479

258.4

5.9

1.75

1.25

Cl H N

H N O

Cl

Musk fragrances Galaxolide 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8hexamethyl-cyclopenta-(γ)-2benzopyran (HHCB) CAS 1222-05-5

Cl Cl

O

Tonalide 7-acetyl-1,1,3,4,4,6-hexamethyl1,2,3,4-tetrahydronaphthalene (AHTN) CAS 1506-02-1

O

258.4

5.7

Traseolide 5-acetyl-1,1,2,6-tetramethyl-3isopropyl-indane (ATII) CAS 68140-48-7

O

258.4

6.3

244.4

5.4

244.4

5.9

206.3

5.9

297.3

4.8

O

Celestolide 4-acetyl-1,1-dimethyl-6tert-butyllindane (ADBI) CAS 13171-00-1

O

Phantolide 6-acetyl-1,1,2,3,3,5 hexamethyllindane (AHMI) CAS 15323-35-0

O

Cashmeran 6,7-dihydro-1,1,2,3,3-pentamethyl4(5H)-indanone (DPMI) CAS 33704-61-9

Musk xylene 2,4,6-trinitro-1,3-dimethyl5-tert-butylbenzene (MX) CAS 81-15-2

O2N

NO2

NO2

414

Water Solubility (mg/L)

O Cl

Triclocarban 3,4,4′-trichloro-carbanilide (TCC) CAS 101-20-2

Molecular Weight

0.49

Table 18.1. Structure and physicochemical parameters of common personal care products. (cont.) Trade Name and CAS Name and Number Musk ketone 4-aceto-3,5-dimethyl-2,6dinitro-tert-butylbenzene (MK) CAS 81-14-1

Structure

O2N

Molecular Weight

Log Kow

294.3

4.3

268.3

5.7

278.3

5.8

266.3

5.9

267.3

4.8

267.3

4.8

264.3

5.1

Water Solubility (mg/L) 1.9

NO2

O Musk ambrette 4-tert-butyl-3-methoxy2,6-dinitrotoluene (MA) CAS 123-69-3

Musk moskene 1,1,3,3,5-pentamethyl4,6-dinitroindan (MM) CAS 145-39-1 Musk tibetene 1-tert-butyl-3,4,5-trimethyl2,6-dinitrobenzene (MT) CAS 145-39-1

2-amino musk xylene 2-amino-4,6-dinitro-1,3-dimethyl5-tert-butylbenzene (2-AMX) CAS 107342-55-2

O O2N

NO2

O2N

NO2

O2N

NO2

O2N

NH2

NO2 4-amino musk xylene 4-amino-2,6-dinitro-1,3-dimethyl5-tert-butylbenzene (4-AMX) CAS 107342-55-6

O2N

NO2

NH2 2-amino musk ketone 4-aceto-2-amino-3,5-dimethyl6-nitro-tert-butylbenzene (2-AMK) CAS 255393-52-3

O2N

NO2

O (continued)

415

Table 18.1. Structure and physicochemical parameters of common personal care products. (cont.) Trade Name and CAS Name and Number Preservatives/antioxidants Methylparaben 4-hydroxybenzoic acid methyl ester (MP) CAS 99-76-3

Structure

O

152.2

O

166.2 O CH2CH3

O

180.2 O CH2CH2CH3

HO O

Butylparaben 4-hydroxybenzoic acid butyl ester (BP) CAS 94-26-8

194.2 O CH2CH2CH2CH3

HO O

Sorbic acid CAS 110-44-1

112.12

4.76

122.12

4.21

3400

228.2

3.79

210

277.4

6.15

OH O

Benzoic acid CAS 65-85-0

OH

430.69

OH

(

O tert-butylhydroquinone (TBHQ) CAS 1948-33-0

Water Solubility (mg/L)

CH3

HO

HO

α-Tocopherol CAS 59-02-9

Log Kow

O

Ethylparaben 4-hydroxybenzoic acid ethyl ester (EP) CAS 120-47-8 Propylparaben 4-hydroxybenzoic acid propyl ester (PP) CAS 94-13-3

Molecular Weight

OH

Sunscreen agents Benzophenone-3 (BP3) CAS 131-57-7

(

166.22

OH

HO

3

O

O O

Octyldimethyl-p-aminobenzoic acid (ODPABA) CAS 21245-02-3

O N

416

2.1

Personal Care Products

417

Table 18.1. Structure and physicochemical parameters of common personal care products. (cont.) Trade Name and CAS Name and Number

Structure

Homosalate (HMS) CAS 118-56-9

O

Molecular Weight

Log Kow

262.3

6.16

290.4

5.8

Water Solubility (mg/L) 20

OH

O

O

Octyl methoxycinnamate (OMC) CAS 5466-77-3

150

O

O O

Avobenzone Butyl methoxydibenoylmethane (BMDBM)

O

310.4

O 4-Methylbenzylidene camphor (4-MBC) CAS 36861-47-9

Octocrylene (OC) CAS 6197-30-4

254.4

4.95

5.1

361.5

7.35

0.2

O

N

O O

triclosan and triclocarban (Table 18.2). For liquid samples (e.g., milk, juice, and soft drinks), solid-phase extraction (SPE) or liquid–liquid extraction (LLE) can be used to extract the two compounds. Solid-phase extraction using Waters Oasis HLB cartridges has been used to extract triclosan and triclocarban from water (Halden and Paull 2005). The target compounds (triclosan and triclocarban) are eluted with organic solvents (4 mL, 50 : 50 methanol/acetone containing 10 mM acetic acid). Deconjugation using β-

glucuronidase/sulfatase (Helix pomatia, H1) is needed for total concentrations (free + conjugated) for milk samples. On-line SPE was also used in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Ye et al. 2008). Milk can also be extracted with hexane using the following procedures (Adolfsson-Erici et al. 2002). The fat extract is redissolved in 2 mL hexane, and triclosan in the extracts is distributed to 2 mL 0.5 M KOH/50% EtOH. The alkaline phase is washed twice with 2 mL hexane and

418

Analysis of Endocrine Disrupting Compounds in Food

Table 18.2. Extraction and analysis of disinfectants and antiseptics. Analytes

Matrix

Sample Preparation

TCS and TCC

Biosolids

PLE with DCM Cleanup using Oasis HLB

TCS and MTCS

Fish and foodstuffs

Matrix solid-phase dispersion (MSPD) Derivatization with MTBSTFA

TCS

Foodstuffs (orange juice, chicken breast, gouda cheese)

Hexane extraction with hand shaking, followed by redissolution with acetonitrile (ACN)

MTCS

Fish

25 g sample was homogenized with 100 g of anhydrous Na2SO4 (dried at 540°C for 12 h) and mixed with 150 mL of solvent (cyclohexane/ DCM, 1 : 1). Extracted twice. GPC cleanup

Determination Technique LC-MS/MS C18 (150 mm × 2.1 mm, 4 μm), mobile phase: MTH and water GC-MS/MS HP-5MS

HPLC-UV and HPLC-MS Kromasil 100 C18 (15 cm × 0.4 cm, 5 μm particle size) Mobile phase: 65% ACN (2 min), to 100% ACN in 15 min GC-EI-MS DB-5 (30 m × 0.25 mm, 1 μm) GC-MS/MS DB-5 (25 m × 0.32 mm)

SIM or MRM

References

TCS: 315 > 162 TCC: 287 > 35 MTCS: 304 > 232, 252, 254 TCS: 347 > 200, 310

Chu and Metcalfe (2007) Canosa et al. (2008)

SanchesSilva et al. (2005)

302 > 252 304 > 252 304 > 254

Balmer et al. (2004)

TCS, triclosan; MTCS, methyl triclosan; TCC, triclocarban.

reextracted into 2 × 3 mL hexane/methyl-tbutyl-ether (90 : 10) after addition of 1 mL 1M HCl. The organic phase is evaporated to dryness, redissolved in 2 mL hexane, and treated with 2 mL H2SO4. After evaporation of the organic phase, 2 mL hexane and 4 mL 0.1 M Na2CO3 is added together with 100 μL acetic anhydride and immediately shaken for 2 min. The organic phase is ready for gas chromatography-mass spectrometry (GC-MS). LLE was also applied to extract triclosan in orange juice samples (Sanches-Silva et al. 2005). In the extraction method, an orange

juice sample (10 g) was weighed in a 40-mL screw-cap centrifuge tube. A 10-mL volume of hexane was added and immediately handshaken for 10 min. Tubes were centrifuged, and the organic phase was removed. Extraction was repeated twice with 10 mL of hexane. The collected hexane phases were combined and evaporated to dryness in a rotary evaporator at 40°C. For solid samples, various extraction techniques are applied, depending on the sample matrix. Chicken breast samples (10 g) were extracted by hand shaking for 10 min with acetonitrile (2 mL) and hexane (10 mL) in

Personal Care Products

40-mL screw-cap centrifuge tubes (SanchesSilva et al. 2005). Extraction was repeated twice with 10 mL hexane. Collected hexane phases were evaporated to dryness in a rotary evaporator at 40°C. However, Gouda cheese was first extracted with 10 mL of hexane three times. Organic phases were pooled and evaporated in a evaporator. The fatty liquid residue obtained was extracted with 2 × 10 mL acetonitrile, and the collected phases were evaporated in a rotary evaporator. The extract was diluted to 10 mL with acetonitrile 90% (v/v) for high-performance liquid chromatography analysis (HPLC). Lipid and other coextracted species for biota and food samples such as fish are an analytical challenge. Multistep cleanup procedures are needed to have a clean extract. Accelerated solvent extraction (ASE, Dionex) can be applied to extract fish samples (Balmer et al. 2004): 5 g of the homogenized fish fillets are mixed with 10 g of Hydromatrix and extracted with cyclohexane/dichloromethane (1 : 1) at room temperature and a pressure of 1500 psi for 9 min (three cycles, with a total solvent volume of about 400 mL). The lipid extracts are then subjected to gel permeation chromatography (GPC) for separation of lipid and analytes. Matrix solid-phase dispersion (MSPD) could also be used for the simultaneous extraction and purification of target species (Canosa et al. 2008). In this technique, samples are first blended and dispersed around the particles of a suitable sorbent using a mortar and pestle and then transferred to a solid-phase extraction (SPE) cartridge, which could also contain a cleanup cosorbent. The efficiency and selectivity of the extraction can be tuned by appropriate selection of washing and elution solvents as well as dispersant and cosorbent materials. For extraction, samples (0.5 g) are mixed with 2 g of sodium sulfate and dispersed in a mortar with a pestle, using 1.5 g of neutral silica and 3 g of SiO2-H2SO4 (10%, w/w), then transferred to a SPE cartridge containing a layer of cosorbent at the bottom. Analytes are eluted with 10 mL of

419

dichloromethane. The extract is evaporated to dryness, using a gentle stream of nitrogen at room temperature, redissolved in 1 mL of ethyl acetate, and filtered.

Musk fragrances Musk fragrances include various classes of natural and synthetic compounds such as polycyclic musks, nitro musks, and amino musks (Table 18.3). For liquid samples such as milk and juices, SPE or LLE can be used to extract the musk fragrances (Ott et al. 1999; Bester 2009). Sequential LLE was applied to extract water samples using npentane and dichloromethane (Ricking et al. 2003). Water samples were first extracted with n-pentane followed by dichloromethane, and then the water samples were acidified to pH 2 and extracted again using dichloromethane. Water can also be extracted using C18 cartridges or C18 disks (Zhang et al. 2008; Bester 2009). The target compounds are eluted with hexane and hexane/dichloromethane (1 : 1, v/v) consecutively. The second fraction (hexane/dichloromethane) is collected and concentrated for GC-MS analysis. Due to their very lipophilic nature, decreased recovery rates are sometimes experienced when using SPE for water samples in comparison to LLE. For solid samples such as fish tissues, Soxhlet or ASE is applied to the extraction using middle polarity solvents (Draisci et al. 1998; Nakata 2005; Zeng et al. 2005; Nakata et al. 2007). Soxhlet extraction was used for marine biological tissues with the following procedures (Nakata 2005). Approximately 1–4 g of tissues are ground with sodium sulfate and extracted with mixed solvents of dichloromethane and hexane (8 : 1) for 7 h using a Soxhlet apparatus. A portion of the extract is used for lipid content measurement by evaporating the solvent until a constant weight is obtained. Lipid in the sample extract is removed by gel permeation chromatography (GPC) with a Bio-beads S-X3 packed

420

Analysis of Endocrine Disrupting Compounds in Food

Table 18.3. Extraction and analysis of musk fragrances. Analytes

Matrix

Sample Preparation

Determination Technique

SIM or MRM

HHCB, AHTN

Marine mammals and sharks

GC-EI-MS DB-1 (30 m × 0.25 mm, 0.25 μm of film thickness)

Polycyclic musks

Fish from the wild

Musk xylene

Rainbow fish and fish oil fish feed

1–4 g of tissues were ground with sodium sulfate and extracted with DCM/ hexane (8 : 1) for 7 h using a Soxhlet apparatus GPC cleanup using a Bio-beads S-X3 Accelerated solvent extraction (ASE) Solvent: ethyl acetate/hexane (1 : 5) 3 g sample mixed with 5 g of Hydromatrix In a cell, insert a cellulose filter and add 5 g alumina and a cellulose again. Homogenization with n-hexane Separation by centrifugation Concentration or Soxhlet extraction for 6 h

HHCB: m/z 243, 258, 213 AHTN: m/z 243, 258, 159 Musk xylene: 282, 297 Musk ketone: 279, 294 Musk ambrett: 253, 268 DPMI: m/z 191, 135, 163, 206 ABDI: m/z 229, 244 AHMI: m/z 229, 187, 244 HHCB: m/z 243, 213, 258 ATII: m/z 215, 173, 258 AHTN: m/z 243, 159, 187, 258 m/z 282

GC-MS Supelcowax 10 (30 m × 0.20 mm, 0.2 μm)

GC-MS HP-5 (30 m × 0.25 mm)

References Nakata (2005)

Draisci et al. (1998)

Fernandez et al. (1996)

DPMI, Cashmeran; ADBI, celestolide; AHMI, phantolide; HHCB, galaxolide; ATII: traseolide; AHTN, tonalide.

glass column (380 mm × 22 mm I.D.). A mixture of 50% hexane in dichloromethane (DCM) is used as the mobile phase at a flow rate of 5 mL/min. The first 60 mL of eluate is discarded, and the following 80 mL fraction, which contains musks, is collected. The eluted solvent is concentrated and passed through a 1.5-g activated silica gel-packed glass column for further fractionation. The first fraction that eluted with 60 mL of hexane is discarded in order to remove some interfering compounds in the next fraction. The second fraction, eluting with 60 mL of 30% DCM in hexane and containing all synthetic fragrances, is collected for GC-MS. Extraction of fish samples can also be carried out by ASE using a mixture of ethyl acetate/hexane (5 : 1, v/v) as the extrac-

tion solvent (Draisci et al. 1998). To obtain a fat-free extract, 5 g of alumina, first activated at 500°C for 4 h and then deactivated with 15% (w/w) water prior to use, is placed at the outlet end of the ASE cell during the extraction. The fish tissue sample is homogenized in a Waring blender, and 3 g of the homogenate is mixed with 5 g of Hydromatrix in a mortar with a pestle. The mixture sample is loaded into the ASE cell on top of conditioned alumina and extracted.

Preservatives/antioxidants Sample preparation procedures vary according to sample matrices and analytical techniques (Table 18.4). Gonzalez et al. (1998 and 1999) developed a detailed sample

421

Human milk

Fatty foods

Soft drinks yogurts sauces Foodstuffs

Five parabens

Antioxidants: tert-butyl-4hydroxyanisole 2,6-di-tert-butyl-p hydroxytoluene tert-butylhydroquinone α-tocopherol α-tocopheryl acetate Preservatives: sorbic acid benzoic acid and their esters

Five preservatives: benzoic acid, sorbic acid, parabens

Preservatives: benzioc acid, sorbic acid, methylparaben, propylparaben

Matrix Edible vegetable oil

11 Synthetic antioxidants and preservatives

Analytes

Fatty samples (margarine, oil, fresh cheese, etc.) mixed with 2 mL hexane, extracted with saturated acetonitrile (in n-hexane/2-propanol : ethanol, 2 : 1 : 1) Gentle shaking for 5 min, hexane phase was discarded, the other frozen at −18°C for 1 h and filtered. The filtrate was placed on a flash evaporator furnished with a water bath at <40°C and concentrated to 0.5 mL within 5 min, residue was dissolved at 25 mL of 0.1 M HNO3, a 5-mL aliquot was introduced into SPE system. Novel solid-phase extraction element called “magic chemisober” (MC) Thermal desorption-gas chromatography (TD-GC) 1 g food sample extracted with methanol in a sonicator

0.25 g oil weighed into a 10 mL centrifuge tube; 3 mL acetonitrile saturated with hexane homogenized for 5 min and centrifuged for 5 min; acetonitrile phase collected, extracted twice. 100 μL milk, 290 μL MeOH mixed and centrifuged at 8000 rpm. Collected 250 μL supernatant, add 250 μL formic acid mixed for the online SPE-HPLCMS/MS

Sample Preparation

Table 18.4. Extraction and analysis of preservatives and antioxidants. Determination Technique

HPLC-DAD Supelco C18 (15 cm × 4.6 mm, 5 μm), UV 254 nm Mobile phase: methanol-acetate buffer (pH 4.4) (35 : 65, v/v) for 9 min, then to methanol-acetate buffer (50 : 50, v/v)

GC-FID GC-MS

HPLC-TOF Zorbax Eclipse XDB-C18 column (150 mm × 2.1 mm, 3.5 μm) Mobile phase: acetonitrile /water at 0.2 mL/min LC-MS/MS APPI source Zorbax Ecipe XDB-C8 (150 mm × 4.6 mm, 5 μm) Negative ion mode

SIM or MRM

Methyl paraben: m/z 151 > 92 Ethyl paraben: m/z 165 > 92 Propyl paraben: m/z 179 > 92 Butyl paraben: m/z 193 > 92 Benzyl paraben: m/z 227 > 92 Chromatogr A 848, 529-536

Molecular ions

References

Saad et al. (2005)

Wang et al. (2006)

Gonzalez et al. (1999)

Ye et al. (2008)

Li et al. (2009)

422

Analysis of Endocrine Disrupting Compounds in Food

pretreatment procedure for GC-FID and GC-MS. Briefly, liquid samples such as soft drinks are degassed in an ultrasonic bath. An accurately weighed amount of 1 g is spiked with an internal standard (2-tert-butyl-4methylphenol) and diluted in 25 mL of 0.1 M HNO3. A 5-mL aliquot of the diluted sample is then directly inserted into the continuousflow SPE system. Solid samples such as skim yogurts, jams, and sauces are first homogenized by magnetic stirring. Portions of 1-g samples and spiked internal standard are placed in a separating funnel and mixed with 25 mL of 10−3 M HCl and 2 mL of saturated potassium perchlorate. The aqueous phase is extracted twice with 10 mL of ethyl ether for 5 min, and the collected ethyl ether is pooled (20 mL) and evaporated to dryness. The residue is dissolved in 25 mL of 0.1 M HNO3 and a 5-mL aliquot is introduced into the SPE system. The extracts are further cleaned up by using an XAD-2 column. For fatty foods, a few grams of fatty sample (margarine, oil, fresh cheese, mayonnaise, or pate) are mixed with 2 mL of saturated hexane (in acetonitrile) at international standards. The suspension of the sample is then extracted with 10 mL of the saturated acetonitrile (in hexane)/2-propanol/ ethanol (2 : 1 : 1) with gentle shaking for 5 min. The hexane phase is discarded and the other frozen at −18°C for 1 h and then filtered. This procedure can remove at least 95% of triglyceride in the food sample. The filtrate is placed on a flash evaporator furnished with a water bath at 40°C and concentrated to approximately 0.5 mL within 5 min. The residue is dissolved in 25 mL of 0.1 M HNO3, and a 5-mL aliquot is introduced into the SPE system for further purification. For HPLC-UV and LC-MS analysis, relatively simpler extraction methods may be used (Saad et al. 2005; Li et al. 2009). Solid food samples (jams, dried fruits, and canned vegetables) are finely ground prior to the extraction. About 1 g sample is accurately weighed in a screw-cap test tube. Twenty-five milliliters of methanol is added and placed in

a sonicator that is maintained at 50°C for 30 min. The test tube is subjected to vortex mixing for 2 min. The contents are filtered through a 0.45-μm nylon-membrane filter, and the clear filtrate is injected into the HPLC column (Saad et al. 2005). For edible oils, about 0.25 g of edible vegetable oil is weighed into a 10-mL glass centrifuge tube, and 3 mL acetonitrile saturated with hexane is added. The mixture is homogenized for 5 min at 1800 rpm with a minishaker. It is then centrifuged for 5 min at 4000 rpm. The acetonitrile phase is collected, and the oil phase is subsequently extracted twice in this way by acetonitrile. All the acetonitrile phases are combined into a 10-mL capacity flask, diluted to the mark with acetonitrile, and analyzed by liquid chromatography coupled with time-offlight mass spectrometry (LC-TOF-MS) (Li et al. 2009). Recently, novel sample preparation techniques such as solid-phase microextraction (SPME), stir bar sorptive extraction (SBSE), and magic chemisorber (MC) have been developed and applied for analysis of various chemicals, including preservatives (Wang et al. 2006). For BHA see Chapter 20.

Sunscreen agents Few studies on sunscreen agents (UV filters) in foods have been reported (Table 18.5), but analytical methods developed for sunscreen agents in environmental matrices can be adopted for the determination of sunscreen agents in foods (Peck, 2006). Sunscreen agents in water can be extracted by LLE using cyclohexane or SPE using C18, Oasis HLB cartridges, or C18 disks. The analytes are recovered from the SPE cartridges using methanol/dichloromethane, and the extracts are further cleaned on silica minicolumns using ethyl acetate (Balmer et al. 2005). SPME has also been used to recover octyldimethyl-p-aminobenzoic acid and benzophenone-3 from water. Sunscreen agents in milk samples were extracted from

Personal Care Products

423

Table 18.5. Extraction and analysis of sunscreen agents. Analytes

Matrix

Sample Preparation

Determination Technique

SIM or MRM

References

4-methylbenzylidene camphor (4-MBC) octocrylene (OC)

Fish

GC-MS

4-MBC: m/z 254.17, 239.14 OC: m/z 249.08, 361.20

Buser et al. (2006)

4-methylbenzylidene camphor (4-MBC) benzophenone-3 (BP3) ethylhexyl methoxycinnamate (EHMC) octocrylene (OC)

Fish

10–25 g of fillets suspended in 100 mL of water in a blender; extracted in a separation funnel with a mixture of dipotassium oxalate (2 mL, 35%), ethanol (100 mL), diethyl ether (50 mL), and n-pentane (70 mL); GPC cleanup Fish samples were homogenized with Na2SO4 and column extracted with DCM/ cyclohexane (1 : 1); GPC cleanup

GC-MS

4-MBC: m/z 254.17, 239.14 BP-3: m/z 228.08, 227.08 EHMC: m/z 178.06, 290.19 OC: m/z 249.08, 361.20

Balmer et al. (2005)

the cream first using sodium sulfate and the solvent n-hexane/acetone (1 : 1), followed by dichloromethane/acetone (1 : 1) (Schlumpf et al. 2008b). Following evaporation of the solvent in a rotary evaporator, the extract was redissolved in cyclohexane/ethyl acetate (1 : 1). To remove lipid, GPC was performed on Bio-beads S-X3 with cyclohexane/ethyl acetate as an eluting solvent. For fish samples, column extraction or ASE has been used in the extraction of some organic UV filters such as 4-methylbenzylidene (4-MBC), benzophenone-3 (BP3), ethylhexyl methoxycinnamate (EHMC), and octocrylene (OC) (Balmer et al. 2005). The fish samples were homogenized with Na2SO4 and column extracted with dichloromethane/cyclohexane (1 : 1). The extracts were cleaned by GPC using a Biobeads S-X3 column and dichloromethane/ cyclohexane (35 : 65) as a mobile phase, followed by silica chromatography.

Buser et al. (2006) used a different extraction method for UV filters in fish. About 10–25 g of fish filet was suspended in 100 mL of ultrapure water and pureed with a hand blender. The fine suspension was transferred quantitatively into one 2-L separation funnel, fortified with internal standards, and extracted by shaking for 1 min each time with a mixture of dipotassium oxalate (2 mL, 35%), ethanol (100 mL), diethyl ether (50 mL), and npentane (70 mL). The lipid content was determined gravimetrically. Following extraction, lipids were removed by GPC using Bio-beads S-X3 (30 × 2.5 cm column) and elution with 5 mL/min of a 1 : 1 mixture of cyclohexane/ ethyl acetate. Solvents were evaporated to 1 mL. Isooctane (2 mL) and hexane (5 mL) were added, and the volume was reduced again to 2 mL. Further cleanup was performed on silica gel columns (5 g plus 1 g of Na2SO4 on top of the column). After conditioning (5 mL) and sample loading, the silica gel

424

Analysis of Endocrine Disrupting Compounds in Food

column was eluted with 35 mL of hexane (fraction 1), then with 40 mL of a 1 : 1 mixture of hexane/dichloromethane (fraction 2) and 40 mL of dichloromethane (fraction 3). UV filters were contained in fractions 2 and 3. More information on BP-3 can be found in Chapter 20.

Analysis Disinfectants/antiseptics Triclosan can be analyzed by GC-MS, GC-MS/MS, LC-MS, or LC-MS/MS, whereas triclocarban can be analyzed by LC-MS or LC-MS/MS. Triclosan needs to be derivatized before injecting it onto GC-MS or GC-MS/MS using various reagents such as bis(trimethylsilyl)trifluoroacetamide (BSTFA), N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) (Ying and Kookana 2007), Nmethyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) (Canosa et al. 2008), acetic anhydride (Adolfsson-Erici et al. 2002), and pentafluorobenzoyl chloride (PFBOCl) (Zhao et al. 2009), whereas methyl triclosan can be directly analyzed by GC-MS (Balmer et al. 2004). The characteristic ions for methyl triclosan are m/z 304, 267, 252, and 232. During the silylation reaction, the hydroxyl group of triclosan is converted into its corresponding trimethylsilyl (tms) ether with MSTFA (or BSTFA) or tbutyldimethylsilyl (tbdms) ethers with MTBSTFA. The characteristic ions used for quantification and confirmation of triclosan are m/z 345, 347, 360, and 362 for its tms derivative and m/z 347, 310, and 200 for its tbdms derivative. The ions for the ethylated derivative of triclosan are m/z 288, 290, 330, and 332 under electron impact mode (EI), whereas those for the pentafluorobenzyl derivative are m/z 482, 482, 287, and 289 under negative chemical ionization mode (NCI). NCI mode of GC-MS produces a very low detection limit of 0.2 ng/L for 1 L of water (Zhao et al. 2009).

Triclosan and triclocarban can be directly analyzed by HPLC-UV, HPLC-MS, or LC-MS/MS (Sanches-Silva et al. 2005; Chu and Metcalfe 2007; Halden and Paull 2005). Triclosan was separated on a Kromasil 100 C18 column (15 cm × 0.4 cm ID, 5 μm particle size) with a mobile phase as follows: 65% to 100% acetonitrile within 15 min at a flow rate of 1 mL/min (Sanches-Silva et al. 2005). UV detection of triclosan is subject to interference, whereas HPLC-MS or LC-MS/MS provides better selectivity and sensitivity. Selected ion monitoring (SIM) at m/z 287 for triclosan and m/z 313 for triclocarban was performed after separation on a C18 column (150 × 2.1 mm, 5 μm) with an isocratic mobile phase of 70% acetonitrile and 30% water with 10 mM acetic acid at 0.2 mL/min (Halden and Paull 2005). The MS/MS is one of the best detectors in the analysis of chemicals in complex sample matrices (Figure 18.1). Triclosan and triclocarban were analyzed in negative ion mode of electrospray ionization (ESI) with multiple reaction monitoring (MRM): m/z 315 > 162 for triclocarban and m/z 287 > 35 for triclosan (Chu and Metcalfe 2007). In this method, chromatographic separation was performed with a mobile phase of water and methanol, but triclosan and triclocarban were poorly resolved. An acidic mobile phase was reported by Halden and Paull (2005), but acid addition would greatly reduce the sensitivity. A mobile phase with 10 mM ammonium acetate may improve chromatographic resolution slightly but at the expense of reduced sensitivity.

Musk fragrances GC-MS with EI mode is routinely used for detection of all musk fragrances (Figure 18.2). Usually, three mass fragments can be detected so that verification and quantification can be performed at each run (Table 18.6). However, the fragmentation pattern usually is dominated by cleavage of methyl groups, giving not very significant fragments

Personal Care Products

425

Figure 18.1. Mass chromatograms of triclosan and triclocarban by liquid chromatography-tandem mass spectrometry in the negative electrospray ionization mode.

MS10 ZXY4501 100

Scan EI+ TIC 4.83e6

21.35

27.90 27.20

23.00 14.64 %

0

12.00

14.00

16.00

18.00

20.00

22.00

24.00

26.00

28.00

30.00

32.00

34.00

Time

Figure 18.2. Total ion chromatogram of polycyclic musks by gas chromatography-mass spectrometry under electron impact mode. Peak 1 (14.64 min): DPMI, 1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentamethyl-4H-inden-4-one; peak 2 (21.35 min): ADBI, 4-acetyl-1,1-dimethyl-6-tert-butylindan; peak 3 (23.00 min): AHMI, 6-acetyl-1,1,2,3,3,5hexamethylindan; peak 4 (27.20 min): coeluting peak, ATII, 5-acetyl-1,1,2,6-tetramethyl-3- isopropylindan, and HHCB, 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-benzopyran; and peak 5 (27.90 min): AHTN, 7-acetyl-1,1,3,4,4,6–hexamethyl-1,2,3,4-tetrahydronaphthalene.

(Bester 2009; Osemwengie and Steinberg 2001; Ricking et al. 2003). Ion trap MS has also been used for analysis of musk fragrances to improve sensitivity and selectivity (Herren and Berset 2000). For amino metabolites, all MS spectra exhibit a peak showing a 30 mass unit loss from the molecular peak, indicating that one nitro group has been reduced. Moreover, among the amino metabolites, one group has the molecular ion (M+) as a base peak as for amino musk ambrette (AMA), amino musk tibetene (AMT), and amino musk ketone (AMK), and the other group has the M+-CH3 fragment ion as base peak as for amino musk moskene (AMM) and amino

musk xylene (AMX). The MS/MS spectra of AMA, AMT, and AMK show a strong, common M+-73 fragment ion (AMA: m/z 165; AMT: m/z 163; and AMK: m/z 191) due to the cleavage of the tertiary butyl group and amino group (M+-57-16). The MS/MS spectra of AMX and AMM show fragment ions at m/z 235 and m/z 216 corresponding to the cleavage of a CH3 and an OH group.

Preservatives/antioxidants A variety of analytical methods have been reported for determining preservatives and antioxidants (Gonzalez et al. 1999; Saad et al.

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Analysis of Endocrine Disrupting Compounds in Food

Table 18.6. Characteristic fragment ions of common personal care products. Compound Disinfectants Triclosan (TCS)

GC-EI-MS

LC-MS(/MS)

345, 347, 360 for its silyl derivative

315 > 162

Triclocarban (TCC)

287 > 35

Reference Ying and Kookana (2007) Chu and Metcalfe (2007) Chu and Metcalfe (2007)

Musk fragrances Galaxolide (HHCB) Tonalide (AHTN) Traseolide (ATII) Celestolide (ADBI) Phantolide (AHMI) Cashmeran (DPMI) Musk xylene (MX) Musk ketone (MK) Musk ambrette (MA) Musk moskene (MM) Musk tibetene (MT) 2-Amino musk xylene (2-AMX) 4-Amino musk xylene (4-AMX) 2-Amino musk ketone (2-AMK)

243, 258, 213, 244 243, 258, 244, 201 215, 216, 173 229, 244, 173, 230 229, 244, 187, 230 191, 192, 135, 206 282, 297 279, 294 253, 268, 254 263, 278, 264 251, 266, 252 267, 252, 218 252, 267, 218 264, 249, 215

Ricking et al. (2003) Ricking et al. (2003) Ricking et al. (2003) Ricking et al. (2003) Ricking et al. (2003) Ricking et al. (2003) Ricking et al. (2003) Ricking et al. (2003) Osemwengie and Steinberg (2001) Osemwengie and Steinberg (2001) Osemwengie and Steinberg (2001) Osemwengie and Steinberg (2001) Osemwengie and Steinberg (2001) Osemwengie and Steinberg (2001)

Preservatives/antioxidants Methylparaben (MP) Ethylparaben (EP) Propylparaben (PP) Sorbic acid Benzoic acid α-Tocopherol tert-Butylhydroquinone (TBHQ)

121, 93, 152 122, 137, 166 121, 138, 180 97, 67, 112 105, 77, 122 430, 165, 205 151, 123, 166

Gonzalez et al. 1999 Gonzalez et al. 1999 Gonzalez et al. 1999 Gonzalez et al. 1999 Gonzalez et al. 1999 Gonzalez et al. 1999 Gonzalez et al. 1999

227, 151, 105 165, 277, 148

Diaz-Cruz et al. (2008) Diaz-Cruz et al. (2008)

120, 262, 138

Diaz-Cruz et al. (2008)

178, 290, 161

Diaz-Cruz et al. (2008)

135, 310, 295 254, 239, 211, 128

Diaz-Cruz et al. (2008) Diaz-Cruz et al. (2008)

204, 360, 249, 232

Diaz-Cruz et al. (2008)

Sunscreen agents Benzophenone-3 (BP3) Octyldimethyl-p-aminobenzoic acid (ODPABA) Homosalate (HMS) CAS 118-56-9 Octyl methoxycinnamate (OMC) Avobenzone (BMDBM) 4-Methylbenzylidene camphor (4-MBC) Octocrylene (OC)

2005; Wang et al. 2006; Li et al. 2009). HPLC-UV is often preferred for the determination of food preservatives and antioxidants. GC-FID or GC-MS with or without derivatization is also employed for selective determination of these compounds; MS is an especially sensitive and selective technique and is gradually gaining ground for analysis. A reverse-phase HPLC method has been successfully applied for the separation and simultaneous determination of the preserva-

tives benzoic acid (BA), sorbic acid (SA), methylparaben (MP), and propylparaben (PP), in 67 foodstuffs (soft drinks, jams, sauces, canned fruits/vegetables) (Saad et al. 2005). The separation is achieved on a Supelco 516 C18 column (150 mm × 4.6 mm, 5 μm) using the mobile phase from methanolacetate buffer (pH 4.4) (35 : 65, v/v) for 9 min, after which it is changed to methanol-acetate buffer (pH 4.4) (50 : 50, v/v). The UV wavelength is set at 230 nm for BA and 254 nm for

Personal Care Products

the others. LC-MS is increasingly used in the determination of antioxidants and preservatives in foodstuffs due to its sensitivity and selectivity. Li et al. (2009) used LC-TOF-MS in negative ionization mode for accurate qualitative and quantitative analysis of food additives, including eight antioxidants and three preservatives in foods. Analysis by LCTOF-MS is accomplished with the accurate mass of the deprotonated molecules [M-H]−, along with the accurate mass of their main fragmention. GC-FID and GC-MS have been performed in the simultaneous determination of 10 additives, tert-butyl-4-hydroxyanisole, 2,6-di -tert-butyl-p-hydroxytoluene, tert-butylhydroquinone, α-tocopherol and α-tocopheryl acetate, sorbic acid, benzoic acid, and their esters (methyl p-hydroxybenzoic acid, ethyl p-hydroxy benzoic acid, and propyl phydroxy benzoic acid) in fatty foods without derivatization (Gonzalez et al. 1999). Three characteristic ions are selected to be monitored in GC-MS analysis: m/z 97, 67, 112 for sorbic acid; m/z 105, 77, 122 for benzoic acid; m/z 121, 93, 152 for methyl-p-hydroxybenzoic acid; m/z 122, 137, and 166 for ethylp-hydroxybenzoic acid; m/z 121, 138, and 180 for propyl-p-hydroxybenzoic acid; m/z 151, 123, and 166 for tert-butylhydroquinone; m/z 430, 165, and 205 for α-tocopherol; and m/z 430, 166, and 472 for α-tocopheryl acetate.

Sunscreen agents GC techniques are preferred for most of the sunscreen agents, whereas GC-EI-MS is very sensitive in the SIM mode (Table 18.6). Characteristic ions are selected for quantification and confirmation: m/z 254.17 and 239.14 for 4-MBC, m/z 228.08 and 227.08 for BP3, m/z 178.06 and 290.19 for EHMC, and m/z 249.08 and 361.20 for OC (Balmer et al. 2005). Some sunscreen agents such as octyl traizone, avobenzone, 4-isopropyldibenzoylmethane, and 2-phenylbenzimidazole-5-

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sulfonic acid are not amenable to GC analysis; they can be analyzed by HPLC-UV and LC-MS or GC-MS after derivatization (DiazCruz et al. 2008).

References Adolfsson-Erici, M., Pettersson, M., Parkkonen, J., Sturve, J. 2002. Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden. Chemosphere 46, 1485–1489. Alslev, B., Korsgaard, B., Bjerregaard, P. 2005. Estrogenicity of butylparaben in rainbow trout (Oncorhynchus mykiss) exposed via food and water. Aquatic Toxicology 72, 295–304. Balmer, M.E., Buser, H.R., Miller, M.D., Poiger, T. 2005. Occurrence of some organic UV filters in wastewater, in surface waters, and in fish from Swiss lakes. Environmental Science and Technology 39, 953–962. Balmer, M.E., Poiger, T., Droz, C., Romanin, K., Bergqvist, P.A., Muller, M.D., Buser, H.-R. 2004. Occurrence of methyl triclosan, a transformation product of the bactericide triclosan, in fish from various lakes in Switzerland. Environmental Science and Technology 38, 390–395. Bester, K. 2009. Analysis of musk fragrances in environmental samples. Journal of Chromatography A 1216, 470–480. Buser, H.R., Balmer, M.E., Schmid, P., Kohler, M. 2006. Occurrence of UV filters 4-methylbenzylidene camphor and octocrylene in fish from various Swiss rivers with inputs from wastewater treatment plants. Environmental Science and Technology 40, 1427–1431. Canosa, P., Rodriguez, I., Rubi, E., Ramil, M., Cela, R. 2008. Simplified sample preparation method for triclosan and methyltriclosan determination in biota and foodstuff samples. Journal of Chromatogrpahy A 1188, 132–139. Chen, J., Ahn, K.C., Gee, N.A., Ahmed, M.I., Duleba, A.J., Zhao, L., Gee, S.J., Hammock, B.D., Lasley, B.L. 2008. Triclocarban enhances testosterone action: A new type of endocrine disruptor? Endocrinology 149 (3), 1173–1179. Chu, S., Metcalfe, C.D. 2007. Simultaneous determination of triclocarban and triclosan in municipal biosolids by liquid chromatography tandem mass spectrometry. Journal of Chromatography A 1164, 212–218. Darbre, P.D. 2006. Environmental oestrogens, cosmetics and breast cancer. Best Practice & Research Clinical Endocrinology & Metabolism 20 (1), 121–143. Daughton, C.G., Ternes, T.A. 1999. Pharmaceuticals and personal care products in the environment: Agents of subtle change? Environmental Health Perspectives 107 (supplement 6), 907–938. Diaz-Cruz, M.S., Llorca, M., Barcelo, D. 2008. Organic UV filters and their photodegradates, metabolites and disinfection by-products in the aquatic environment. Trends in Analytical Chemistry 27, 873–887.

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Draisci, R., Marchiafava, C., Ferretti, E., Palleschi, L., Catellani, G., Anastasio, A. 1998. Evaluation of musk contamination of freshwater fish in Italy by accelerated solvent extraction and gas chromatography with mass spectrometric detection. Journal of Chromatography A 814, 187–197. Fernandez, C., Carballo, M., Tarazona, J.V. 1996. A new method to determine musk xylene in water sewages fish and related products. Chemosphere 32, 1805– 1811. Gonzalez, M., Gallego, M., Valcarcel, M. 1998. Simultaneous gas chromatographic determination of food preservatives following solid-phase extraction. Journal of Chromatography A 823, 321–329. Gonzalez, M., Gallego, M., Valcarcel, M. 1999. Gas chromatographic flow method for the preconcentration and simultaneous determination of antioxidant and preservative additives in fatty foods. Journal of Chromatography A 848, 529–536. Halden, R.U., Paull, D.H. 2005. Co-occurrence of triclocarban and triclosan in U.S. water resources. Environmental Science and Technology 39, 1420– 1426. Herren, D., Berset, J.D. 2000. Nitro musks, nitro musk amino metabolites and polycyclic musks in sewage sludges–quantitative determination by HRGC-ion -trap-MS/MS and mass spectral characterization of the amino metabolites. Chemosphere 40, 565–574. Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., Buxton, H.T. 2002. Pharmaceuticals, hormones and other organic wastewater contaminants in U.S. streams, 1999–2000: A national reconnaissance. Environmental Science and Technology 36, 1202–1211. Li, X.Q., Ji, C., Sun, Y.Y., Yang, M.L., Chu, X.G. 2009. Analysis of synthetic antioxidants and preservatives in edible vegetable oil by HPLC/TOF-MS. Food Chemistry 113, 692–700. Nakata, H. 2005. Occurrence of synthetic musk fragrances in marine mammals and sharks from Japanese coastal waters. Environmental Science and Technology 39, 3430–3434. Nakata, H., Sasaki, H., Takemura, A., Yoshioka, M., Tanabe, S., Kannan, K. 2007. Bioaccumulation, temporal trend, and geographical distribution of synthetic musks in the marine environment. Environmental Science and Technology 41, 2216–2222. Osemwengie, L.I., Steinberg, S. 2001. On-site solidphase extraction and laboratory analysis of ultratrace synthetic musks in municipal sewage effluent using gas chromatography-mass spectrometry in the full-scan mode. Journal of Chromatography A 932, 107–118. Ott, M., Failing, K., Lang, U., Schubring, C., Gent, H.J., Georgii, S., Brunn, H. 1999. Contamination of human milk in Middle Hesse, Germany—a cross-sectional study on the changing levels of chlorinated pesticides, PCB congeners and recent levels of nitro musks. Chemosphere 38, 13–32. Peck, A.M. 2006. Analytical methods for the determination of persistent ingredients of personal care products in environmental matrices. Analytical and Bioanalytical Chemistry 386, 907–939.

Ricking, M., Schwarzbauer, J., Hellou, J., Svenson, A., Zitko, V. 2003. Polycyclic aromatic musk compounds in sewage treatment plant effluents of Canada and Sweden–first results. Marine Pollution Bulletin 46, 410–417. Saad, B., Bari, M.F., Saleh, M.I., Ahmad, K., Talib, M.K.M. 2005. Simultaneous determination of preservatives (benzoic acid, sorbic acid, methylparaben and propylparaben) in foodstuffs using high-performance liquid chromatography. Journal of Chromatography A 1073, 393–397. Sanches-Silva, A., Sendon-Garcia, R., Lopez-Hernandez, J., Paseiro-Losada, P. 2005. Determination of triclosan in foodstuffs. Journal of Separation Science 28, 65–72. Schlumpf, M., Durrer, S., Faass, O., Ehnes, C., Fuetsch, M., Gaille, C., Henseler, M., Hofkamp, L., Maerkel, K., Reolon, S., Timms, B., Tresguerres, J.A.F., Lichtensteiger, W. 2008a. Developmental toxicity of UV filters and environmental exposure: A review. International Journal of Andrology 31, 144–151. Schlumpf, M., Kypke, K., Vokt, C.C., Birchler, M., Durrer, S., Faass, O., Ehnes, C., Fuetsch, M., Gaille, C., Henseler, M., Hofkamp, L., Maerkel, K., Reolon, S., Zenker, A., Timms, B., Tresguerres, J.A.F., Lichtensteiger, W. 2008b. Endocrine active UV filters: Developmental toxicity and exposure through breast milk. Chimia 62, 345–351. Wang, L., Zhang, X., Wang, Y., Wang, W. 2006. Simultaneous determination of preservatives in soft drinks, yogurts and sauces by a novel solidphase extraction element and thermal desorption-gas chromatography. Analytica Chimica Acta 577, 62–67. Yang, L.H., Ying, G.G., Su, H.C., Stauber, J.L., Adams, M.S., Binet, M.T. 2008. Growth-inhibiting effects of 12 antibacterial agents and their mixtures on the freshwater microalga (Pseudokirchneriella subcapitata). Environmental Toxicology and Chemistry 27, 1201–1208. Ye, X., Bishop, A.M., Needham, L.L., Calafat, A.M. 2008. Automated on-line column-switching HPLCMS/MS method with peak focusing for measuring parabens, triclosan, and other environmental phenols in human milk. Analytica Chimica Acta 622, 150–156. Ying, G.G., Kookana, R.S. 2007. Triclosan in wastewaters and biosolids from Australian wastewater treatment plants. Environment International 33, 199–205. Zeng, X., Sheng, G., Xiong, Y., Fu, J. 2005. Determination of polycyclic musks in sewage sludge from Guangdong, China using GC-EI-MS. Chemosphere 60, 817–823. Zhang, X., Yao, Y., Zeng, X., Qian, G., Wu, M., Sheng, G., Fu, J. 2008. Synthetic musks in the aquatic environment and personal care products in Shanghai, China. Chemosphere 72, 1553–1558. Zhao, J.L., Ying, G.G., Wang, L., Yang, J.F., Yang, X.B., Yang, L.H., Li, X. 2009. Determination of phenolic endocrine disrupting chemicals and acidic pharmaceuticals in surface water of the Pearl Rivers in South China by gas chromatography-negative chemical ionization-mass spectrometry. Science of the Total Environment 407, 962–974.

Chapter 19 Polycyclic Aromatic Hydrocarbons Peter Šimko

Polycyclic aromatic hydrocarbons One of the most important groups of chemicals that are actually harmful to human health is the group of polycyclic aromatic hydrocarbons (PAHs). They are characterized by two or more condensed aromatic rings in a molecular structure and have a strong lipophilic character. PAHs are formed during the thermal decomposition of organic mass such as fuel, wood, and coal, especially at limited access of oxygen in the range of 500°C– 900°C (Bartle 1991). Alternatively, they are also contained in crude oil, and during accidents they enter directly into the environment and become direct sources of pollution. The temperature of smoke formation plays a decisive role because the amounts of PAH contained in smoke increase linearly with the temperature of smoke generation in the interval of 400°C–1000°C (Tóth and Blaas 1972). In the prehistoric past, humans probably hung their catch over the fire to protect it from scavengers, and from this time smoking was widely used not only for production of smoked products with a special organoleptic profile but also for preservation effects by inactivating enzymes and microorganisms. So far, techniques of smoking have gradually improved, and various procedures have been developed in different regions for treating meat, cheese, and fish. Today, the technology is used mainly for enrichment of foods with

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

specific characteristics (taste, odor, and appearance) that are popular in the market (Šimko 2005). However, PAHs are also formed upon direct thermal meat treatments such as roasting, grilling, and frying. Fat is the main source of hydrocarbons, and formation of PAHs during charcoal broiling is directly dependent on the fat content of the meat. Melted fat from heated meat that drips onto the hot coals is thermally decomposed, giving rise to the formation of PAHs, which are then deposited on the meat surface as the smoke rises (Larsson et al. 1983). The seeddrying processes using direct firing for production of hot air can be responsible for major PAH contamination of some vegetable oils, such as coconut and grapeseed oils. Apart form the formation itself, the temperature also affects the structure and number of PAHs. The number of PAHs present in smoked fish can reach 100 compounds (Grimmer and Böhnke 1975) that have different effects on living organisms.

Behavior of PAHs in an organism According to current knowledge, some PAHs are able to interact with enzymes (such as aryl hydrocarbon hydroxylases) in organisms to form PAH dihydrodiol derivates. These reactive products (called bay region dihydrodiol epoxides) are believed to be ultimate carcinogens that are able to form covalently bounded adducts with proteins and nucleic acids. In general, DNA adducts are thought to initiate cell mutation that results in a malignancy (Bartle 1991). A direct mutagenic potential of 429

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Analysis of Endocrine Disrupting Compounds in Food

14 PAHs and PAHs containing fractions isolated from smoked and charcoal-broiled samples was studied using the Ames test and focused on strains TA 98 and TA 100. The most potential mutagenicity was observed on PAH fractions isolated from smoked fish, treated before smoking with nitrites in an acid solution (Kangsadalampai et al. 1997). To simplify an interpretation of real risk of PAHs to human health, there have been attempts to express objectively the real risk using toxic equivalency factors (Nisbet and La Goy 1992). However, this approach does not reflect wider aspects of a potential toxicity of oxidized PAH products due to the effect of ultraviolet light, as well as other environmental factors (Law et al. 2002). Moreover, PAH content in contaminated foods can be affected not only by environmental factors but also by diffusion processes into or from plastic packaging materials (Šimko et al. 1995; Šimko 2005). PAHs are suspected endocrine disruptors, together with such compounds as zearalenone, dioxins, polychlorinated biphenyls, furans, phenols, and several pesticides (most prominent being organochlorine insecticides such as endosulfan, atrazine, DDT, and its derivatives), xenoestrogens, and phytoestrogens. Frouin et al. (2007) observed a dysfunction in steroid synthesis in the bivalve Mya arenaria after exposure to PAHs. Gesto et al. (2009) found that PAHs can disrupt the activity of pineal organs of teleost fish, quantifying the diurnal and nocturnal pineal content of some indoles and methoxyindoles, including melatonin. Although increase of melatonin levels could play a relevant role as a toxicityprotection factor, this disruption could be a threat for the survival of the animals in their natural environment.

Legislative aspects and international normalization of PAHs in foods and food additives With regard to the harmful effects of PAHs on living organisms, some European countries have enacted maximum limits of these com-

pounds in smoked meat products at different levels. To simplify such problems associated with the variability of PAH composition, benzo[a]pyrene (BaP) had generally been accepted as the indicator of total PAH presence in smoked foods, despite the fact that BaP constitutes only between 1% and 20% of the total carcinogenic PAHs (Andelman and Suess 1970). As of 2010, the European Union (EU) has unified by adopting the European Commission (EC) Regulation 1881/2006 limiting BaP content in various foods. Limits for individual groups of food products are shown in Table 19.1. Table 19.1. Maximum tolerable levels of BaP in various foods Foodstuffs

Oils and fats (excluding cocoa butter) intended for direct human consumption or use as an ingredient in foods. Smoked meats and smoked meat products. Muscle meat of smoked fish and smoked fishery products, excluding bivalve mollusks. The maximum level applies to smoked crustaceans, excluding the brown meat of crab and excluding the head and thorax meat of lobster and similar large crustaceans (Nephropidae and Palinuridae). Muscle meat of fish other than smoked fish. Crustaceans, cephalopods, other than smoked. The maximum level applies to crustaceans, excluding the brown meat of crab and excluding the head and thorax meat of lobster and similar large crustaceans (Nephropidae and Palinuridae). Bivalve mollusks. Processed cereal-based foods and baby foods for infants and young children. Infant formulas and follow-on formula, including infant milk and follow-on milk. Dietary foods for special medical purposes intended specifically for infants.

Maximum Levels (μg kg−1 wet wt.) 2.0

5.0 5.0

2.0 5.0

10.0 1.0 1.0 1.0

Polycyclic Aromatic Hydrocarbons

Apart from this, the EC has also adopted either the directive 2005/10/EC laying down the sampling methods and the methods of analysis for the official control of BaP levels in foodstuffs or the recommendation 2005/108/EC on the further investigation into the levels of PAHs in certain foods, such as benzo[a]anthracene (BaA), benzo[b]fluoranthene (BbF), benzo[j]fluoranthene (BjF), benzo[k]fluoranthene (BkF), benzo[g,h,i] perylene (BghiP), chrysene (Chr), BaP, cyclopenta[c,d]pyrene (CcpP), dibenzo[a,h] anthracene (DahA), dibenzo[a,e]pyrene (DaeP), dibenzo[a,h]pyrene (DahP), dibenzo[a,i]pyrene (DaiP),dibenzo[a,l]pyrene(DalP),indeno[1,2,3cd]pyrene (IcdP), and 5-methylchrysene. The Joint Expert Committee for Contaminants and Additives (JECFA) of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) has defined another compound, benzo[c]fluorene (BcF), which should also be monitored with regard to its effects on living organisms. As for liquid smoke flavors (LSF), the EC has adopted regulation 2065/2003 relating to the production of smoke flavorings intended to be used for food flavoring. This regulation has limited the maximum acceptable concentrations of BaP at 10 μg kg−1 and BaA at 20 μg kg−1 in these products. Finally, directive 88/388/EEC has limited the maximum residual levels of BaP at 0.03 μg kg−1 in foodstuffs flavored by LSF. For international trade purposes, the Joint Expert Committee for Food Additives and Contaminants of FAO and WHO has adopted a specification that tolerates the concentration in liquid smoke flavors at the levels of 10 μg kg−1 for BaP, and 20 μg kg−1 for BaA, (Report of the Joint FAO/ WHO Expert Commission 1987).

Sample preparation In general, composition of food matrices is the most important factor regarding sample treatment to remove all compounds able to interfere with the analyte. For this, different procedures for sample pretreatment are

431

undertaken to reach the highest recoveries of analytes as possible.

Sample treatment of smoked meat From an analytical point of view, meat and its products belong to problematic matrices with regard to the presence of various interfering compounds. Moreover, PAHs as lipophile compounds have the tendency to diffuse not only into the nonpolar part of the sample but also inside tissue cells due to the existing concentration gradient. For this reason a simple solvent extraction with nonpolar solvent seems to be insufficient to reach high recovery. Grimmer and Böhnke (1975) isolated PAHs from smoked fish and smokedried cobra with boiling methanol prior to sample hydrolysis with methanolic KOH. It was found that only about 30% BaP and other PAHs were extractable from the samples, whereas an additional alkaline hydrolysis of meat protein yielded another 60% of PAHs. It was concluded that PAHs were linked adsorptively to high molecular structures not destroyed with boiling methanol. Although more than 80% of the methanol used could be recovered, this contained only one-third of the PAHs contained in the samples. As postulated, alkaline hydrolysis with aqueous methanolic KOH is an absolute necessity to isolate PAHs quantitatively from such types of samples. Alkaline hydrolysis usually takes 2–4 h, depending on the character of the sample. Lean tissues take less time than adipose- and collagencontaining tissues. This sample treatment was adopted by following many experimental works (Fretheim 1976; Binnemann 1979; Larsson 1982; Lawrence and Weber 1984). On the other hand, Vassilaros et al. (1982) observed that the use of an alcohol is superfluous and contributes to interference problems in the final analysis because of methyl ester formation from fatty acids and methanol that are then difficult to remove from the PAH fraction. Takatsuki et al. (1985) found that during alkaline hydrolysis BaP may be

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Analysis of Endocrine Disrupting Compounds in Food

partially decomposed by the coexistence of alkaline conditions, light oxygen, and peroxides in aged ethyl ether. They proposed using amber glass, adding Na2S as an antioxidant, distilling the ethyl ether just before use, and preventing air from contacting the adsorbents. To protect PAHs from light decomposition, Karl and Leinemann (1996) used brown glassware carefully rinsed with acetone before using an alkaline hydrolysis. Even so, some authors also recommended direct extraction with organic solvents. Potthast and Eigner (1975) proposed a procedure based on the mixing of preground sample with chloroform and anhydrous Na2SO4 to remove water from the extract. After adding Celite, the fat portion became uniformly distributed over the surface of the adsorbent. Although the authors achieved a recovery of 95%–100% of BaP added at a level of 10 μg, there is a real assumption that they recovered only “free” PAHs accessible with solvent. This procedure also was used in the work of Alonge (1988). Cejpek et al. (1995) tested the efficiency of several organic solvents to obtain the fat portion from meat samples. The most efficient solvent was a mixture of chloroform and methanol (2 : 1), less effective was chloroform, and the worst yields were achieved with methanol. This confirms the observations of Grimmer and Böhnke (1975) regarding insufficient capability of methanol to extract quantitatively PAHs from meat samples. Otherwise, the use of a chloroform–methanol mixture, also called the Folch agent, is widely used in food analysis for extraction of lipids when methanol makes possible extraction of lipids from inside of cells by denaturation of cell wall proteins. Joe et al. (1984) digested samples of smoked food with KOH and extracted PAHs with Freon 113 (1,1,2-trichloro1,2,2-trifluoroethane). Chen et al. (1996) compared efficiency of extraction from freezedried samples when sonication and Soxhlet procedures were employed. Recovery studies showed that Soxhlet extraction was only suitable prior to the sonication method. An accel-

erated procedure of extraction was tested by Wang et al. (1999). Samples were extracted in a Dionex extractor as well as a Soxhlet apparatus. Advanced solvent extraction (ASE) technique was found to be comparable with or even better than the reference Soxhlet method when significant reductions in time of extraction and solvent consumption were achieved. García-Falcón et al. (2000) accelerated extraction of PAHs from freeze-dried samples into hexane with microwave treatment and hexane extract, then saponified them with ethanolic KOH.

Sample treatment of cheese Cheese matrix is very similar to meat matrix regarding constituents such as fat, proteins, and water. This means that destruction of bulk matrix is carried out in KOH at elevated temperature, followed by extraction into nonpolar solvents such as cyclohexane, concentration, and washing on a solid-phase extraction column (Aniello et al. 2004).

Sample treatment of oils The three procedures most commonly used for treatment of oils are liquid–liquid partition following the scheme of Grimmer and Böhnke (1975), caffeine complexation, and saponification (Weisshaar 2002). With the partition method, the oil sample is dissolved in an organic solvent such as cyclohexane, and PAHs are extracted with a solution of dimethylformamide-water mixture or dimethyl sulfoxide; most of the lipidic matter (mainly constituted of triglycerides) remains in the organic phase. Isolation is performed by dilution with water to change the coefficients of partition of the PAHs between the two phases and to back-partition into cyclohexane. This procedure allows reducing the mass of the residue to 10% of the initial value. A relatively rapid method for determining PAHs in fats and oils uses the phenomenon of caffeine–PAH complex formation. The

Polycyclic Aromatic Hydrocarbons

sample is dissolved in cyclohexane, and the PAHs are selectively extracted by means of vigorous shaking in a caffeine–formic acid solution. After decomposing the complex with an aqueous sodium chloride solution (2%), the PAHs are extracted with cyclohexane. The saponification method makes it possible to recover PAHs from matrices with cyclohexane extraction after 40 min of saponification under reflux using alcoholic KOH (Moret and Conte 2000).

433

SFE was mixed with alumina and extracted PAHs were concentrated in an octadecylsilane trap. In both cases, 91% recoveries of BaP spiked at 15 ng were found and no statistically significant differences were observed. Taking into account the expense of the SFE extractor, they recommended using a simple SPE procedure. Guillén et al. (2000a, b) alkalized LSF with methanolic KOH and heated it under reflux for 3 h, followed by extraction of PAHs into dichlormethane or cyclohexane.

Sample treatment of liquid smoke flavors

Preseparation procedures

Sample treatment of liquid smoke flavor (LSF) matrix is different from the treatment of processed meats due to easy access of organic solvent “inside” a liquid matrix. There is not usually any reason to treat these samples by time-consuming hydrolysis under reflux. A different situation could arise when LSF is in a solid state (e.g., applied on starch, gelatin, or encapsulated). Despite this, some authors preferred alkaline hydrolysis of LSF under reflux. However, adding KOH is strongly recommended to transform phenols to polar, nonextractable phenolates prior to PAH extraction with nonpolar solvent. White et al. (1971) alkalized water-soluble LSF and resinous condensates that settled out of LSF after storage with KOH solution and extracted PAHs into isooctane. Silvester (1980) extracted PAHs from alkalized liquid LSF with hexane. Radecki et al. (1978) alkalized LSF with ethanolic KOH solution and maintained it at 60°C for 30 min prior to the extraction into cyclohexane. After alkalization, a direct extraction of PAH with cyclohexane was used by Šimko et al. (1992). On the other hand, Gomaa et al. (1993) saponified LSF with methanolic KOH for 3 h and than extracted PAHs into cyclohexane. Laffon Lage et al. (1997) used the solid-phase extraction (SPE) technique on Sep Pak C18 for PAH isolation and compared it to (supercritical fluid extraction) SFE when the sample for

At this point, procedures are more or less the same for processed meats and food additives. But sometimes, mainly after adipose tissue hydrolysis, the presence of lipoproteins in nonpolar solvent requires their removal prior to preseparation with one-step liquid– liquid partition between nonpolar and polar solvent (e.g., hexane–water/dimethylforamid [Grimmer and Böhnke 1975], methanol/ water, or dimethylsulfoxide/water-cyclohexane [Lawrence and Weber 1984; Karl and Leinemann 1996]); or two-step liquid–liquid partition (e.g., NaCl/water and DMF/water [Vaessen et al. 1988]); or precipitation of lipoproteins with Na2WO4 [Šimko 1991; Šimko et al. 1991; Šimko et al. 1993]). For preseparation, deactivated Florisil (Šimko et al. 1991; Lawrence and Weber 1984; Wang et al. 1999; Gomaa et al. 1993; Guillén et al. 2000a; Šimko et al. 1993; Mottier et al. 2000; Stijve and Hischenhuber 1987); silica gel (Larsson 1982; Takatsuki et al. 1985; Mottier et al. 2000); alumina (Vaessen et al. 1988); and Celite (White et al. 1971; Silvester 1980) are used frequently. The only study (Silvester 1980) reported that elution of BaP from Florisil and silica gel with hexane was impossible, and for this reason alumina was recommended for preseparation of concentrated PAH extracts. Guillén et al. (2000b) preferred elution of silica with cyclohexane prior to Florisil dichlormetane elution to

434

Analysis of Endocrine Disrupting Compounds in Food

obtain higher recoveries with reduced amounts of interfering substances, which were eluted from Florisil with dichlormethane. Another effective preseparation procedure is GPC (gel permeation chromatography) on Sephadex LH 20 (Takatsuki et al. 1985) or BioBeads S-X3 (Cejpek et al. 1995). Mottier et al. (2000) cleaned concentrated cyclohexane extracts by SPE, using conditioned isolute aminopropyl and C18 columns. Two different techniques were applied when cyclohexane extract was first cleaned with (GPC) on Sephadex LH 20 and on silica gel (Vaessen et al. 1988). The last procedure is also possible to carry out in reverse mode (Afolabi et al. 1983). In all cases, removal of organic solvents by vacuum evaporation to concentrate PAHs is an unavoidable operation. This may be a critical step, particularly if it is presumed that light PAHs such as fluorene (Flu), antracene (Ant), and phenanthrene (Phe) are present in the extracts. In such cases, organic solvents should not be evaporated to dryness because these PAHs could be lost due their volatility. This cautious manipulation is not necessary if only PAHs with boiling points above 370°C are determined (Grimmer and Böhnke 1975).

Thin-layer chromatography Thin-layer chromatography (TLC) is an older analytical method used for determination of PAHs in various matrices. Haenni (1968) discussed the development of analytical tools for control of PAHs in food additives and in food by the use of ultraviolet specification within specific wavelength ranges. For this, Schaad (1970) reviewed various chromatographic separation procedures, including TLC. White et al. (1971) used two systems for PAH separation. The first consisted of 20% N, N-dimethylformamide in ethyl ether as a stationary phase and isooctane as a mobile phase. Fluorescent spots were scraped out from the cellulose layer and eluted with hot methanol. After concentration, the sample

was developed in the second system, using ethanol/toluene/water (17 : 4 : 4) as developer. Fluorescent spots were eluted again from the cellulose acetate layer, and the ultraviolet spectrum was recorded against isooctane in a reference cell. The observed maxima were compared with those in the spectra of known PAHs obtained under the same instrumental conditions. Estimation of quantity of the identified compounds was made by the baseline technique in conjunction with spectra of these PAHs, and the identification was confirmed by spectrophotofluorometry. This method has become the basis of the Association of Analytical Communities (AOAC) Official Method 973.30, adopted in 1974 (AOAC 1998).

Gas chromatography Today, gas chromatography (GC) is widely used for the determination of PAHs in food analysis. Determination of the large number of PAHs in samples requires columns with high efficiency. To separate some critical pairs as well as isomers of methyl derivatives of certain PAHs, capillary columns (50 m × 0.3–0.5 mm) that can achieve 50,000– 70,000 high equivalent theoretical plate (HETP) are especially convenient. However, packed columns used for determination of PAH (Grimmer and Böhnke 1975) had lower HETP, ranging between 20,000 and 30,000, and for this reason they were not suitable for quantity determination. Two stationary phases, OV-17 and OV-101, were used for separation of BaP from BeP, DajA from DahA, and Phe from Ant. Successful separation of Chr from BaA was achieved using OV-17 stationary phase, but separation of BbF, BjF, and BkF isomers on packed columns was not possible (Grimmer and Böhnke 1975). Radecki et al. (1978) tested various stationary phases (GE SE 30, OV-1, SE-52, OV-7, OV-101, BMBT, BBBT) on Chromosorb W, Chromosorb W HP, Gas Chrom, and Diatomite CQ supports in packed

Polycyclic Aromatic Hydrocarbons

columns to develop a precise GC method for assaying BaP in LSF. However, separation of BaP from BeP and Per was not possible using SE 30, OV-1, SE-52, OV-7, or OV-101 stationary phases. Nematic phases gave a good separation of BaP from its isomers, but they were not suitable for analysis due to their poor thermal stability. Detection of PAH is not a serious problem because a response of the flame ionization detector (FID) is practically equal for all compounds and is linear over a large concentration range (about 1–1.106), according to the carbon content. However, the use of FID is sometimes hampered by the need for very thorough cleanup procedures, with the accompanying risk of severe losses and possible misidentification (Tuominen et al. 1986). A mass spectrometry detector (MSD) has also been used successfully for PAH analysis in many cases (Lee et al. 1981). The use of an MSD operating in selected ion-monitoring mode makes it possible to simplify the timeconsuming cleanup procedure (Tuominen et al. 1986), and it is recommended especially for quantitative analysis. The ion trap detector (ITD) has some advantages over a traditional MSD. The ITD uses electric fields to hold the ions within the ion storage regions. The ITD is then scanned through the mass range, causing the ions to be ejected from this region sequentially, from low to high mass. The ejected ions are detected by a conventional electron multiplier. Thus, the characteristic of the ITD is that ionization and mass analysis take place in the same space. This contrasts with a conventional MSD, which requires a separate ionization source, focusing lenses, and analyzer, which is associated with low mechanical tolerances (Williams et al. 1988). Sometimes, separation of isomers is a quite serious problem even though capillary columns are used. Dennis et al. (1984) were not able to separate BjF from BkF. Speer et al. (1990) were not able to separate Chr from triphenylene (Tph); BbF, BjF, and BkF from each other; and

435

DahA from DacA. Problems associated with separation of Chr from Tph are also reported in works of Guillén et al. (2000a, b). Wise et al. (1993) discussed difficulties in separating BbF from BkF isomers. On the other hand, Chen and Chen (2005) separated BbF and BkF sufficiently on DB-1 fused silica capillary column. Jira (2004) separated all 16 European priority PAH compounds, including problematic BbF, BjF, and BkF isomers using GC column VF 17 ms. A review of preseparation procedures as well as GC conditions used for the determination of PAH in foods are summarized in Table 19.2.

High-pressure liquid chromatography In recent years, the high-pressure liquid chromatography (HPLC) method has been used intensively to determine the presence of PAH in food, as reported by Bartle (1991), Tamakawa (2004), and Stahl and Eisenbrand (1988). Formerly used stationary phases such as alumina and silica gel were later replaced with chemically bonded phases, particularly reverse phases such as ODS, widely used at the time. For determination of PAH in food, Hunt et al. (1977) developed a phtalimidopropylsilane (PPS) stationary phase and compared it with octadecylsilane (ODS). They found that the PPS column was able to separate BkF from Per, which was impossible by ODS column. HPLC has some advantages in PAH analysis, as follows (Tamakawa, 2004): • Separation of isomers shows very good resolution. • There is sufficient sensitivity and specificity of ultraviolet fluorescence detection. • Molecular sizes of PAHs can be estimated on the basis of retention time using a reversed-phase column. • Compounds with high molecular weight can be determined. • Analyses are usually carried out at ambient temperature, with no risk of thermal decomposition of analytes

436

Smoked fish

Smoked fish, smoked meat spreads

Smoked fish and fish products

Smoked meat products

Smoked sausages

Smoked fish

Smoked fish

Barbecued sausages

Sample Saponification with mixture of ethanol, water, and KOH, extraction with cyclohexane, preseparation by SPE on isolute aminopropyl and C18 columns Extraction with pentane, precleaning on silica gel, and Sephadex LH-20 Saponification in methanolic KOH, liquid–liquid extraction (methanol-watercyclohexane and DMF-water-cyclohexane) and GPC on Sephadex LH 20 Saponification in methanolic KOH, liquid–liquid extraction (methanol-watercyclohexane and DMF-watercyclohexane), precleaning on silica gel, and GPC on Sephadex LH 20 Saponification with mixture of methanol, water, and KOH, partition with DMF, precleaning on Kiesel gel 60 Saponification in methanolic KOH, liquid–liquid extraction (methanol-watercyclohexane and DMF-watercyclohexane), precleaning by CC on silica gel, and GPC on Sephadex LH 20 Saponification with mixture of methanol, water, and KOH, extraction with cyclohexane, cleaning up on Florisil, partitioning with DMSO-hexane Saponification with methanol-water-KOH mixture under reflux, extraction into cyclohexane, extraction of PAHs with caffeine/formic acid, washing with NaCl solution, extraction into cyclohexane, preseparation on silica gel

Sample Treatment and Preseparation

240°C, isothermal 165°C for 6 min, 165 → 255°C at 4°C/min

25°C → 180°C rapidly → 320°C at 8 °C/min 110°C, isothermal for 1.5 min → 210°C at 30°C/ min → 290°C at 3°C/ min → 300°C at 10°C/min

25 m × 0.28 mm capillary column/SE-54 55 m × 0.3 mm glass capillary column/SE-54

30 m × 0.25 mm capillary column/DB-5 30 m × 0.25 mm capillary fused silica column/DB-5

MSD

FID, MSD

FID

MSD

FID

FID MSD

1. 120 → 250°C, 1°C/min 2. 250°C, isothermal 260°C, isothermal

MSD

100 → 260°C, 3°C/min

Detection MSD

25 m × 0.2 mm quartz capillary column/SE-54 10 m × 2 mm packed columns/5% OV-101 and OV-17 on sorbent Gas Chrom 10 m × 2 mm packed column/5% OV-101 on sorbent Gas Chrom

Temperature Program 80°C for 0.5 min → 230°C at 8°C/min → 300°C at 5°C/min

25 m × 0.2 mm capillary column/SPB-5

Column/Stationary Phase

Table 19.2. Preseparation procedures and GC conditions used for determination of PAHs in foods. References

Karl and Leinemann 1996

Lawrence and Weber 1984

Larsson 1982

Binnemann 1979

Fretheim 1976

Afolabi et al. 1983 Grimmer and Böhnke 1975

Mottier et al. 2000

437

Heating with methanolic KOH under reflux, extraction with cyclohexane, cleaning up by SPE technique on Florisil Heating with methanolic KOH under reflux, extraction with cyclohexane, cleaning up by SPE technique on LC silica Saponification with methanolic KOH, extraction with cyclohexane, partition with DMF-water, cleanup on silica gel and with GPC on Bio-beads S-X3 Extraction with methanol in Soxhlet app., + KOH, extraction into n-hexane, cleanup on Sep-Pak Florisil cartridge Accelerated solvent extraction, gel permeation washing procedure, solidphase extraction

LSF

Smoked products

Smoked chicken

Smoked meats

LSF

Alkalization with KOH solution, extraction with cyclohexane, cleanup on silica

LSF

Sample Treatment and Preseparation Direct solvent extraction (ASE), cleanup on Florisil

Smoked salmon, sausages, pork

Sample

TR-50 MS column (10 m × 0.1 mm × 0.1 μm)

30 m × 0.32 mm/DB-5

70°C isothermal for 1 min → 150°C at 10°C/min → 280 °C at 4°C/ min, hold for 14 min 140°C isothermal for 1 min, at 10°C/min → 240°C, at 5°C/min → 270°C, at 30°C/min → 280°C, at 4°C/min → 290°C, at 30°C/min → 315°C and at 3°C/min → 330°C

50°C isothermal for 0.5 min → 130°C at 8°C/ min → 290°C at 5°C/min 50°C isothermal for 0.5 min → 130°C at 8°C/ min → 290°C at 5°C/min 70°C → 280°C at 5°C/min

50°C isothermal for 0.5 min → 180°C at 30°C/ min → 300°C at 7°C/min

Temperature Program 40°C isothermal for 1 min → 250°C at 12°C/min → 310°C at 5°C/min

Column/Stationary Phase 30 m × 0.25 mm capillary column/cross-linked 5% phenyl methyl siloxane HP-5 MS 25 m × 0.2 mm fused silica capillary column/HP-5 cross linked with 5% phenylmethylsilicone 60 m × 0.25 mm fused silica capillary column/HP-5 MS, 5% phenyl methyl siloxane 60 m × 0.25 mm fused silica capillary column/HP-5MS, 5% phenyl methyl siloxane 50 m capillary column/DB-5

Detection

References

Chiu et al. 1997 Djinovic et al. 2008a; Djinovic et al. 2008b

HRMS

Speer et al. 1990

Guillén et al. 2000b

Guillén et al. 2000a

Šimko et al. 1992

Wang et al. 1999

ITD

MSD

MSD

MSD

MSD

MSD

438

Analysis of Endocrine Disrupting Compounds in Food

HPLC equipped with MSD is an effective tool for characterization of high-molecular, thermally unstable compounds; for example, BaP metabolites were identified and determined by this method in microbore mode (Bieri and Greaves 1987). Due to high absorption of the light in the UV part of the spectrum and intensive fluorescence, both types of detectors are able to reliably detect concentrations at the microgram per kilogram level. On the other hand, measurements by nonspecific detection systems, particularly optical detectors, though often precise, can also be much less accurate due to possible chemical interferences not chromatographically resolved or otherwise avoided prior to the measurement. The major impurities in the PAH fractions appear to be alkylated PAHs, which have very similar responses in optical detection systems to their unsubstituted analogs (Sim et al. 1987). Regarding a diode array detector, confirmation of peak purity and identification is possible, but due to the broad absorption bands in UV spectra it is highly probable that there will be some interference if one particular wavelength is chosen for quantification. In any case, identification must be based on retention time. The fluorescence detector provides very high selectivity and sensitivity, particularly for those with excitation and emission wavelengths that can by varied throughout the analyses. However, fluorescence suffers from not being able to provide “broad spectrum” analyses (i.e., a wide variety of compounds) because of the presence of alkylated PAH compounds. A review of preseparation procedures as well as HPLC conditions used for determination of PAH in foods are summarized in Table 19.3.

Comparison of gas chromatography and high-pressure liquid chromatography In many reports, also mentioned here, authors have compared advantages and drawbacks of GC and HPLC, with studies especially aimed

at recovery, quality of separation processes, time of analysis, price of equipment, etc. Dennis et al. (1984) compared results of analysis of two smoked foods obtained by GC and HPLC. Thirty-five pairs of analyses were tested using statistical procedures (student t-test). From these, twenty-five were not significantly different within the 95% confidence limits employed. But data for BkF/benzofluorantenes and DahA/ dibenzoanthracenes were not compared because different analytes were measured. Standard deviations indicated that repeatability of both methods was very good, usually within 10%, and gave comparable data throughout a wide range (0.2–1000 μg kg−1). In the conclusion of this study, it was stressed that capillary GC possessed a much greater resolving power, in terms of plate number, so that many more PAHs can be separated and determined. On the other hand, HPLC was able to separate individual isomers (BbF and BkF, Chr, and Tph); that is, it had greater selectivity. Chiu et al. (1997) compared separation and detection conditions of both methods analyzing smoked chicken. They found that 16 of the priority PAH pollutants as set by the Environmental Protection Agency (EPA) can be simultaneously separated by HPLC using a gradient solvent system and detection by FLD (fluorescence detection) with a setting of seven for the programmable wavelength. With GC, a temperature programming method, it is also possible to resolve these 16 PAHs. The presence of impurities in smoked meat products can interfere with the identification and quantification of PAHs by HPLC. With ITD, the PAHs can be identified, even in the presence of fat impurities. The retention times by HPLC were shorter than those by GC, and HPLC had better separation for most compounds than did GC. Sim et al. (1987) compared GC and HPLC methods analyzing 16 PAH pollutants. As pointed out, the chromatographic resolution may be divided into a combination of column capacity, column efficiency, and

439

Alkalization with NaOH solution, extraction with hexane, cleanup on alumina

Alkalization with ethanolic and aqueous NaOH, 30 cm × 4 mm, extraction into cyclohexane, partitioning with μBondapak C18/ Corasil DMSO-water, extraction into cyclohexane LSF: Saponification with methanolic KOH, 25 cm × 4.6 mm, extraction into cyclohexane, purification on Supelcosil LC-PAH Florisil Meat products: digestion with KOH solution, extraction with Freon 113, purification on Florisil

LSF

LSF

LSF, smoked food products

A: methanol/acetonitrile/water 50 : 25 : 25, B: acetonitrile; 1 min 100% A, 25th min 100% B.

15 cm × 4.6 mm Supelcosil LC PAH, 5 μm

25 cm × 4.6 mm Partisil 10 ODS

Acetonitrile/water 60 : 40 for 5 min then 100% of acetonitrile in 15 min, hold for 15 min, then decrease to 60% for 10 min

Methanol/water 7 : 3, 2 mL min−1

I. Acetonitrile/water 7 : 3, isocratic, 2 mL min−1 II. Acetonitrile/water 40 : 60, gradient to 100% acetonitrile within 25 min III. Acetonitrile/water 55 : 45, gradient to 100% acetonitrile within 23 min Methanol/acetonitrile/water 35 : 35 : 30, isocratic

Acetonitrile/water 7 : 3 for 1 min then gradient linearly up to 9 : 1 in 19th min, then to 100% acetonitrile from 20 to 40 min then isocratic for 55 min

ET 15 cm × 4 mm, Nucleosil 5 C10 PAH

12.5 cm × 4.6 mm Envirosep-pp C18 5 μm

Acetonitrile/water 8 : 2, isocratic, 1 mL min−1

Radial-Pak PAH

Extraction with methanol in Soxhlet app., + KOH, extraction into n-hexane, cleanup on Pep-Pak Florisil

Smoked sausage, smoked meat

Smoked fish

Fish, shellfish

Smoked frankfurters, smoked meats

Acetonitrile/water 7 : 3, isocratic, 3 mL min−1

25 cm × 4.6 mm, RP-18, 5 μm

Saponification with mixture of methanol, water, and KOH, extraction with cyclohexane, cleanup on Florisil, partitioning with DMSO/ hexane Saponification with methanol-water-KOH mixture under reflux, extraction into n-hexane, cleanup on silica gel Saponification with methanol-water-KOH mixture under reflux, extraction into cyclohexane, extraction of PAHs with caffeine/formic acid, washing with NaCl solution, extraction into cyclohexane, preseparation on silica gel Extraction with chloroform/methanol mixture, preseparation by GPC on Bio-beads S-X3

Smoked fish, smoked meat spreads

Acetonitrile/water 3 : 1, isocratic, 1.5 mL min−1

Saponification with mixture of methanol, water, and KOH, extraction with cyclohexane, washing with Na2WO4 solution, cleanup on Florisil

30 cm × 3 mm, Separon SGX C18 RP, 5 μm,

Mobile Phase Acetonitrile/water 8 : 2, isocratic, 1.5 mL min−1

Column/Stationary Phase 25 cm × 4 mm Lichrosorb RP 18

Smoked meat products

Sample Treatment and Preseparation Saponification with ethanolic KOH, extraction into cyclohexane, washing with saturate NaCl solution, cleanup on silica gel

LSF, smoked meats

Sample

Table 19.3. Preseparation procedures and HPLC conditions used for determination of PAH in foods. Detection

FLD Ex/Em 254/375 nm

FLD Variable Ex (240–293) Em (340–498) nm UVD 230–360 nm FLD Variable Ex (232–302) Em (330–484) nm FLD Ex/Em 280/390 nm UVD 280 nm

UVD 254 nm FLD Ex/Em 250/370 nm FLD Ex/Em 370/410 nm UVD 240, 254, 260 nm FLD Ex/Em 300/408 and 280/395 nm

FLD Ex: 305, 381 nm Em: 389, 430, 520 nm FLD Ex/Em 310/410 nm

References

(continued)

Gomaa et al. 1993

Radecki et al. 1978

Silvester 1980

Chen et al. 1996

Cejpek et al. 1995

Karl and Leinemann, 1996

Vassilaros et al. 1982

Šimko 1991; Šimko et al. 1991; Šimko et al. 1992; Šimko et al. 1993 Lawrence and Weber 1984

Alonge 1988

440 Saponification with methanolic KOH, extraction with n-hexane, preseparation by SPE on CN-bonded silica Saponification with methanol-water-KOH mixture under reflux, extraction into n-hexane, cleanup on silica gel LSF: Saponification with methanolic KOH, extraction into cyclohexane, purification on Florisil Smoked products: digestion with KOH solution, extraction with Freon 113, purification on Florisil Saponification with methanolic KOH, extraction into cyclohexane, preseparation by SPE on Kiesel gel Extraction with methanol in Soxhlet app., + KOH, extraction into n-hexane, cleanup on Sep-Pak Florisil Dissolution in hexane, cleanup on alumina

Smoked meat products

Smoked meats

Tocopherol concentrates

Smoked chicken

Smoked meat products

LSF, smoked foods

Lyophilisation, microwave-assisted solvent extraction by hexane, cleanup by solid-phase extraction

Saponification with mixture of methanol, water, and KOH, extraction with cyclohexane, partitioning with DMSO-hexane

Smoked fish, ham

Smoked fish

Sample Treatment and Preseparation Direct extraction with chloroform, preseparation on preparation silica column

Sample Smoked fish

Acetonitrile/water 8 : 2, isocratic, 1 mL min−1 A: water; B: methanol/acetonitrile 1 : 1; I. segment: 1 : 80 to 100% B in 20 min II. segment: 100% B for 5 min III segment: 100 to 80 B in 5 min Acetonitrile/water 9 : 1, isocratic, 0.5 mL min−1

15 cm × 6mm, ODS, 5 μm particles 12.5 cm × 4 mm Lichrosphere 100 RP-18

12.5 cm × 4 mm, Chrompack PAH-Säule 12.5 cm × 4.6 mm Envirosep-pp 5 μm C18

C18 reversed-phase (Supelcosil LC-PAH), 250 × 3 mm I.D., 5 l-m particle size

RP Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm I.D., 5 l-m)

Acetonitrile/water 8 : 2, isocratic, 0.5 mL min−1

Acetonitrile/water 55 : 45, gradient to 100% acetonitrile within 23 min 1.2 mL min−1 Acetonitrile (A) water (B) at 1.0 mL/ min. The gradient elution program was as follows: 0 → 13 min, 40% A isocratic; 13 → 22 min, linear gradient 40% → 34% A; 22 → 32 min linear gradient 34% A → 16% A; and finally, back to the initial condition and recondition the column of acetonitrile and water at a flow rate of 1 mL/min. The gradient elution program started with 40% acetonitrile (isocratic for 5 min), going linearly to 100% acetonitrile (total run time: 40 min).

Acetonitrile/water 6 : 4, linearly to 9 : 1 for 35 min

Mobile Phase Preparation column: pentane/5% DCM, 0.8 mL min−1 Analytical column: Water/acetonitrile 6 : 4 for 5 min then to 100% acetonitrile over 40 min, 1.5 mL min−1

Column/Stationary Phase Preparation column: 25 cm × 4.6 mm, silica 5 μm, Analytical column: 15 × 4.6 mm 5 μm particle, Supelcosil LC-PAH Spherisorb ODS 5 μm precolumn and 5 μm VydacODS analytical column Nucleosil 100–5 C 18 PAK

Table 19.3. Preseparation procedures and HPLC conditions used for determination of PAH in foods. (cont.)

FLD Variable nm

FLD Variable nm

FLD Ex/Em 290/430 nm FLD Variable nm

FLD Ex/Em 290/430 nm FLD Ex/Em 370/410 nm FLD Ex/Em 365/418 nm

FLD Ex/Em 290/430 nm

Detection FLD Variable nm

Purcaro et al. 2009

Yang et al. 2008

Chiu et al. 1997

Räuter 1997

Yabiku et al. 1993

Ova and Onaran 1998

Hartmann 2000

Dennis et al. 1984

References Moret et al. 1999

Polycyclic Aromatic Hydrocarbons

separation selectivity. GC has higher column efficiency and thus has an advantage for complex mixture analysis, but HPLC can often have higher column selectivity, which is more suitable for separation of isomeric compounds. Thus both methods should be viewed as complementary in the analysis of

441

PAHs, and they are essential for precise and reliable analysis.

Occurrence of PAHs in foods Immediately after studies showed the carcinogenic effects of PAHs, research workers

Table 19.4. BaP content in foods.

a b

Sample

No. of Analysed/ Positive Samples

Barbecued pork and beef

2/2

Concentration of BaP (μg kg−1) Min. Max. 2.5 4.5

Frankfurters grilled by various ways Charbroiled hamburgers Fried hamburgers Ham, bacon, fish, sausage

5/5 2/2 2/0 19/19

0.1 1.5 — 0.3

212 4.0 — 18

Frankfurters, meat, sausages Ham, pork, meat products Sausages, spread, salami, fish Fermented products, frankfurters Sausages, special products Dark smoked meat products Heavy smoked sausage Cooked out fat of sausage Sausage, pork Ham, bacon Sausage, fish, pork tasso Sausage, poultry, bacon Ribbons, ham, sausages, bacon Mutton meat Salami, bacon Heavy smoked ham products Smoked cured ham, smoked raw sausage Barbecued meat sausages Smoked beef ham, smoked pork ham Smoked beef ham, smoked pork ham, bacon without skin, bacon with skin, cajna sausage and sremska sausage Smoked pork meat

8/8 74/69 17/17 17/7 386 5/5 1/1 1/1 2/2 3/3 5/0 5/3 6/5 5/5 4/4 196/196 18/18

0.1 0.2 0.1 0.05 0.6 17.1 4.8 7.7 0.3 0.2 — 0.1 0.2 0.1 0.2 0.03 0.1

12.0 56.5 9.5 0.15 100 39.9 5.2 0.4 — 0.4 1.3 5.6 0.5 >10.0 0.4

References

Howard and Fazio 1969 Larsson et al. 1983 Lawrence and Weber 1984 Stijve and Hischenhuber 1997 Roda et al. 1999 Potthast 1978 Šimko et al. 1991 Fretheim 1976 Binnemann 1979 Šimko et al. 1989 Šimko et al. 1993 Cejpek et al. 1995 Joe et al. 1984 Wang et al. 1999 Gomaa et al. 1993 Yabiku et al. 1993 Dennis et al. 1984 Lintas et al. 1979 Rauter 1997 Jira 2004

7/2 4/4 6/6

0.3 0.1 0.2

2.8 0.3 1.0

Mottier et al. 2000 Djinovic et al. 2008a Djinovic et al. 2008b

11/11

6.0

35.1

Smoked meat products (sausage, pork, beef, bacon) Cheese

8/4

0.1

0.8

Stumpe-Viksna et al. 2008 Purcaro et al. 2009

144

Vegetable oils Olive pomace oil Olive oil Tocopherol products Tea

7/4

0.4 0.2 0 21.2 0.1 7.7 0

2.1a 0.5b 22 25.2 0.2 469.5 39.7

Smoked traditionally. Flavored by liquid smoke flavors.

3/3 3/3 8/5

Aniello et al. 2004 Moreda el al. 2004 Weisshaar 2002 Perelló et al. 2009 Yang et al. 2008 Lin et al. 2005

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started to find real situations of PAH content in food products. As has been proven, technologically correct smoking processes contaminate food products with PAHs at low levels; usually BaP content is below 1 μg kg−1. Far more dangerous are smoking processes under uncontrolled conditions, typical inhome “wild” smoking when preparing heavy smoked “farm” products and in developing countries without technological knowledge and hygienic control. These products bring a serious and real risk of cancer to the consumer, especially after long-term consumption due to BaP content sporadically reaching up to several hundreds of micrograms per kilogram (Alonge 1987; Šimko 2002; Dobríková and Svétlíková 2007). On the other hand, oils may represent an important source of PAHs due to volume of consumption and frequency of occurrence (Moret and Conte 2000). Moreover, from time to time there are problems with over-the-limit concentrations of PAHs in oils sold on the market. For example, the European Union banned import of sunflower oil from Ukraine in 2009 because the oil was contaminated by PAHs. It was found that the oil was contaminated during transport in a tank car previously used for transporting crude oil. Notably, maybe even alarming, is fact that an important source of PAHs can be tocopherol products. These products are now frequently consumed to improve skin color and its resistance to sunlight. As shown Table 19.4, some may contain considerable concentrations of PAHs, which may be a threat throughout the life of consumer.

Acknowledgement This contribution is the result of the project implementation “Centre of Excellence for Contaminants and Microorganisms in Food” supported by the Research & Development Operational Programme funded by the ERDF.

References Afolabi, O. A., Adesulu, E. A., Oke, O. L. 1983. Polynuclear aromatic hydrocarbons in some Nigerian preserved freshwater fish species. Journal of Agricultural and Food Chemistry, 31:1083–1090. Alonge, D. O. 1987. Factors affecting the quality of smoke-dried meats in Nigeria. Acta Alimentaria, 16:263–270. Alonge, D. O. 1988. Carcinogenic polycyclic aromatic hydrocarbons (PAH) determined in Nigerian kundi (smoke-dried meat). Journal of the Science of Food and Agriculture, 43:167–172. Andelman, J. B. and Suess, M. J. 1970. PAH in the water environment. Bulletin WHO, 43:479–508. Aniello, A., Mercogliano, R., Vollano, L., Pepe, T., Cortesi, M. L. 2004. Levels of benzo[a]pyrene (BaP) in ‘mozzarella di bufala campana’ cheese smoked according to different procedures. Journal of Agricultural and Food Chemistry, 52:4452–4455. Association of Analytical Communities (AOAC). 1998. Official Method 973.30 AOAC International, 16th ed. Arlington, VA. 48–11995. Bartle, K. D. 1991. Analysis and occurrence of polycyclic aromatic hydrocarbons in food. In: Food Contaminants, Sources and Surveillance, C. Purchase and R. Purchase, eds., Cambridge: The Royal Society of Chemistry, pp. 41–60. Bieri, R. H. and Greaves, J. 1987. Characterization of benzo(a)pyrene metabolites by high performance liquid chromatography-mass spectrometry with a direct liquid introduction interface and using negative chemical ionization. Biomedical and Environmental Mass Spectrometry, 14:555–561. Binnemann, P. H. 1979. Benz(a)pyrene in Fleischerzeugnissen. Zeitschrift für Lebensmittel Untersuchung und Forschung, 169:447–452. Cejpek, K., Hajšlová, J., Jehlicˇková, Z., Merhaut, J. 1995. Simplified extraction and cleanup procedure for the determination of PAHs in fatty and protein rich matrices. International Journal of Environmental Analytical Chemistry, 61:65–80. Chen, B. H., Wang, C. Y., Chiu, C. P. 1996. Evaluation of analysis of polycyclic aromatic hydrocarbons in meat products by liquid chromatography. Journal of Agricultural and Food Chemistry, 44:2244–2251. Chen, J. and Chen, S. 2005. Removal of polycyclic aromatic hydrocarbons by low density polyethylene from liquid model and roasted meat. Food Chemistry, 90:461–469. Chiu, C. P., Lin, Y. S., Chen, B. H. 1997. Comparison of GC–MS and HPLC for overcoming matrix interferences in the analysis of PAHs in smoked food. Chromatographia, 44:497–504. Commission Directive 88/388/EEC of 22 June 1988 on the approximation of the laws of the Member States relating to flavourings for use in foodstuffs and to source materials for their production. Official Journal of the European Union, L184, 15.07.1988: 61. Commission Directive 2005/10/EC of 4 February 2005 laying down the sampling methods and the methods

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of analysis for the official control of the levels of benzo(a)pyrene in foodstuffs. Official Journal of the European Union, L34, 8.02.2003:15. Commission Recommendation 2005/108/EC of 4 February 2005 on the further investigation into the levels of polycyclic aromatic hydrocarbons in certain foods. Official Journal of the European Union, L34, 8.02.2003:43. Commission Regulation 2065/2003/EC of 10 November 2003 on smoke flavourings used or intended for use in or on foods. Official Journal of the European Union, L309, 26.11.2003:1. Commission Regulation 1881/2006/EC of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union, L364, 20.12.2006:5. Dennis, M. J., Massey, R. C., McWeeny, D. J., Larsson, B., Eriksson, A., Sahlberg, G. 1984. Comparison of capillary gas chromatographic and high-performance liquid chromatographic method of analysis for polycyclic aromatic hydrocarbons in food. Journal of Chromatography, 285:127–133. Djinovic, J., Popovic, A., Jira W. 2008a. Polycyclic aromatic hydrocarbons (PAHs) in traditional and industrial smoked beef and pork ham from Serbia. European Food Research and Technology, 227:1191–1198. Djinovic, J, Popovic, A., Jira W. 2008b. Polycyclic aromatic hydrocarbons (PAHs) in different types of smoked meat products from Serbia. Meat Science, 80:449–456. Dobríková, E. and Svétlíková, A. 2007. Occurence of benzo[a]pyrene in some foods of animal origin in the Slovak Republic. Journal of Food and Nutrition Research, 46:181–185. Fretheim, K. 1976. Carcinogenic PAH in Norwegian smoked meat sausages. Journal of Agricultural and Food Chemistry, 24:976–979. Frouin, H., Pellerin, J., Fournier, M., Pelletier, E., Richard, P., Pichaud, N., Rouleau, C., Garnerot, F. 2007. Physiological effects of polycyclic aromatic hydrocarbons on soft shell clam (Mya arenaria). Aquatic Toxicology, 82:120–134. García-Falcón, M. S., Simal-Gandara, J., CarrilGonzalez-Barros, S. T. 2000. Analysis of benzo(a) pyrene in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwaveassisted treatment of samples. Food Additives and Contaminants, 17:957–964. Gesto, M., Tintos, A., Rodríguez-Illamola, A., Soengas, J.L., Míguez, J.M. 2009. Effects of naphthalene, βnaphthoflavone and benzo(a)pyrene on the diurnal and nocturnal indoleamine metabolism and melatonin content in the pineal organ of rainbow trout, (Oncorhynchus mykiss). Aquatic Toxicology, 92:1–8. Gomaa, E. A., Gray, I. J., Rabie, S., Lopez-Bote, C., Booren, A. M. 1993. Polycyclic aromatic hydrocarbons in smoked food products and commercial liquid smoke flavourings. Food Additives and Contaminants, 10:503–521. Grimmer, G. and Böhnke, H. 1975. Polycyclic aromatic hydrocarbon profile analysis of high protein foods,

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oils and fats by gas chromatography. Journal of Association of Official Analytical Chemists, 58: 725–733. Guillén, M. D., Sopelana, P., Partearroyo A. 2000a. Determination of polycyclic aromatic hydrocarbons in commercial liquid smoke flavourings of different composition by gas chromatography–mass spectrometry. Journal of Agricultural and Food Chemistry, 48:126–131. Guillén, M. D., Sopelana, P., Partearroyo, A. 2000b. Polycyclic aromatic hydrocarbons in liquid smoke flavorings obtained from different types of wood. Effect of storage in polyethylene flasks on their concentrations. Journal of Agricultural and Food Chemistry, 48:5083–5087. Haenni, E. O. 1968. Analytical control of PAH in food and food additives. Residue Reviews, 24:42–78. Hartmann, K. 2000. Benzo[a]pyren Bestimmung bei mit Räucharomageräucherten Fleischerzeugnissen. Deutsche Lebensmittel Rundschau, 96:163–166. Howard, J. W., Fazio, T. 1969. A review of PAH in foods. Journal of Agricultural and Food Chemistry, 17:527-531. Hunt, D., Wild, P., Crosby, N. T. 1977. A new chemically bonded stationary phase for the determination of polynuclear aromatic hydrocarbons by high pressure liquid chromatography. Journal of Chromatography, 130:320–323. Jira, W. 2004. A GC/MS method for the determination of carcinogenic polycyclic aromatic hydrocarbons (PAH) in smoked meat products and liquid smokes. European Food Research and Technology, 218: 208–212. Joe, F. L., Salemme, J., Fazio, T. 1984. Liquid chromatographic determination of trace residues of polynuclear aromatic hydrocarbons in smoked foods. Journal of Association of Official Analytical Chemists, 67: 1076–1082. Kangsadalampai, K., Butryee, C., Manoonphol, K. 1997. Direct mutagenicity of the polycyclic aromatic hydrocarbon-containing fraction of smoked and charcoal-broiled foods treated with nitrite in acid solution. Food and Chemical Toxicology, 35:213–218. Karl, H. and Leinemann, M. 1996. Determination of polycyclic aromatic hydrocarbons in smoked fishery products from different smoking kilns. Zeitschrift für Lebensmittel Untersuchung und Forschung, 202: 458–464. Laffon Lage, B., Garcia Falcon, S., Gonzalez Amigo, M. S., Lage Yusty, M. A., Simal Lozano, J. 1997. Comparison of supercritical fluid extraction and conventional liquid-solid extraction for the determination of benzo(a)pyrene in water-soluble smoke. Food Additives and Contaminants, 14:469–474. Larsson, B. K. 1982. Polycyclic aromatic hydrocarbons in smoked fish. Zeitschrift für Lebensmittel Untersuchung und Forschung, 174:101–107. Larsson, B. K., Sahlberg, G. P., Eriksson, A. T., Busk, L. A. 1983. Polycyclic aromatic hydrocarbons in grilled food. Journal of Agricultural and Food Chemistry, 31:867–873.

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Law, R. J., Kelly, C., Baker, K., Jones, J., McIntosh, A. D. 2002. Toxic equivalency factors for PAH and their applicability in shellfish pollution monitoring studies. Journal on Environmental Monitoring, 4:383–388. Lawrence, J. F. and Weber, D. F. 1984. Determination of polycyclic aromatic hydrocarbons in Canadian samples of processed vegetable and dairy products by liquid chromatography. Journal of Agricultural and Food Chemistry, 32:789–794. Lee, M. L., Novotny, M.V., Bartle, K. D. 1981. Analytical Chemistry of Polycyclic Aromatic Hydrocarbons. New York: Academic Press. Lin, D., Tu, Y., Zhu L. 2005. Concentrations and health risk of polycyclic aromatic hydrocarbons in tea. Food and Chemical Toxicology, 43:41–48. Lintas, C., De Matthaeis, M. C. 1979. Determination of benzo(a)pyrene in smoked, cooked and toasted food products. Food Cosmetology and Toxicology, 17:325328. Moreda, W., Rodríguez-Acuna, R., del Carmen PérezCamino, M., Arturo, C. 2004. Determination of high molecular mass polycyclic aromatic hydrocarbons in refined olive pomace and other vegetable oils. Journal of the Science of Food and Agriculture, 84:1759–1764. Moret, S., Conte, L., Dean, D. 1999. Assessment of polycyclic aromatic hydrocarbon content of smoked fish by means of a fast HPLC/HPLC method. Journal of Agricultural and Food Chemistry, 47:1367– 1371. Moret, S. and Conte, L. 2000. Polycyclic aromatic hydrocarbons in edible fats and oils: Occurrence and analytical methods. Journal of Chromatography A, 882: 245–253. Mottier, P., Parisod, V., Turesky, R. J. 2000. Quantitative determination of polycyclic aromatic hydrocarbons in barbecued meat sausages by gas chromatography coupled to mass spectrometry. Journal of Agricultural and Food Chemistry, 48:1160–1166. Nisbet, I. C. T. and La Goy, P. K. 1992. Toxic equivalency factors (TEFS) for polycyclic aromatic hydrocarbons. Regulatory Toxicology and Pharmacology, 16:290–300. Ova, G. and Onaran, S. 1998. Polycyclic aromatic hydrocarbons contamination in salmon-trout and eel smoked by two different methods. Advances in Food Sciences, 20:168–172. Perelló, G., Martí-Cid, R., Castell, V., Llobet, J. M., Domingo, J. L. 2009. Concentrations of polybrominated diphenyl ethers, hexachlorobenzene and polycyclic aromatic hydrocarbons in various foodstuffs before and after cooking. Food and Chemical Toxicology, 47:709–715. Potthast, K. 1978. Smoking methods and their effect on the content of 3,4-benzpyrene and other constituents of smoke in smoked meat products. Fleischwirtschaft, 58:371–375. Potthast, K. and Eigner, G. 1975. A new method for the rapid isolation of polycyclic aromatic hydrocarbons from smoked meat products. Journal of Chromatography, 103:173–176. Purcaro, G., Moret, S., Conte, L. S. 2009. Optimisation of microwave assisted extraction (MAE) for polycy-

clic aromatic hydrocarbon (PAH) determination in smoked meat. Meat Science, 81:275–280. Radecki, A., Lamparczyk, H., Grzybowski, J., Halkiewicz, J. 1978. Separation of polycyclic aromatic hydrocarbons and determination of benzo(a) pyrene in liquid smoke preparations. Journal of Chromatography, 150:527–532. Räuter, W. 1997. Content of benzo(a)pyrene in smoked foods. Ernährung, 21:447–448. Roda, A., Simoni, P., Ferri, E.N., Girotti, S., Ius, A., Rauch, P., Poplstein, M., Pospisil, M., Pipek, P., Hochel, I., Fukal, L. 1999. Determination of PAHs in various smoked meat products and different samples by enzyme immunoassay. Journal of the Science of Food and Agriculture, 79:58–62. Schaad, R. 1970. Chromatographie (Karzinogener) Polyzyclicer Aromatischer Kohlenwasserstoffe. Chromatography Review, 13:61–64. Silvester, D. S. 1980. Determination of 3,4-benzpyrene and benzanthracene in phenolic smoke concentrates. Journal of Food Technology, 15:413–420. Sim, P. G., Boyd, R. K., Gershey, R. M., Guevremont, R., Jamieson, W. D., Quilliam, M. A., Gergely, R. J. 1987. A comparison of chromatographic and chromatographic/mass spectrometric techniques for determination of polycyclic aromatic hydrocarbons in marine sediments. Biomedical and Environmental Mass Spectrometry, 14:375–381. Šimko, P., Dubravický, J., Smirnov, V. 1989. Vplyv technológie údenia na množstvo benzo(a)pyrénu v údených mäsových výrobkoch. Potravináˇrske Veˇ dy, 7:59-63. Šimko, P. 1991. Changes of benzo[a]pyrene content in smoked fish during storage. Food Chemistry, 40: 293–300. Šimko, P. 2002. Determination of polycyclic aromatic hydrocarbons in smoked meat products and liquid smoke flavourings by gas chromatography and high pressure liquid chromatography. Journal of Chromatography B, 770:3–18. Šimko, P. 2005. Factors affecting elimination of polycyclic aromatic hydrocarbons in smoked meat foods and liquid smoke flavours. Molecular Nutrition and Food Research, 49:637–647. Šimko, P., Karovicˇová, J., Kubincová, M. 1991. Changes in benzo[a]pyrene content in fermented salami. Zeitschrift für Lebensmittel Untersuchung und Forschung, 193:538–540. Šimko, P., Petrík, J., Karovicˇová, J. 1992. Determination of benzo[a]pyrene in liquid smoke preparations by high pressure liquid chromatography and confirmation by gas chromatography-mass spectrometry. Acta Alimentaria, 21:107–114. Šimko, P., Gergely, Š., Karovicˇová, J., Drdák, M., Knežo, J. 1993. Influence of cooking on benzo[a] pyrene content in smoked sausages. Meat Science, 34:301–309. Šimko, P., Khunová, V., Šimon, P., Hrubá, M. 1995. Kinetics of food contamination with polycyclic aromatic hydrocarbons from contaminated recycled low density polyethylene film. International Journal of Food Science and Technology, 30: 807–812.

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Speer, K., Steeg, E., Horstmann, P., Kuehn, T., Montag, A. 1990. Determination and distribution of polycyclic aromatic hydrocarbons in native vegetable oils, smoked fish products, mussels and oysters, and bream from the river Elbe. Journal of High Resolution Chromatography and Chromatography Communications, 13:104–111. Stahl, W. and Eisenbrand, G. 1988. Determination of polynuclear aromatic hydrocarbons and nitrosamines. In: HPLC in Food Analysis, R. Macrae, ed., London: Academic Press, pp. 377–412. Stijve, T. and Hischenhuber C. 1987. Simplified determination of benzo(a)pyrene and other aromatic hydrocarbons in various food materials by HPLC and TLC. Deutsche Lebensmittel Rundschau, 83:276–282. Stumpe-Viksna, I., Bartkevics, V., Kukare, A., Morozovs, A. 2008. Polycyclic aromatic hydrocarbons in meat smoked with different types of wood. Food Chemistry, 110:794–797. Takatsuki, K., Suzuki, S., Sato, N., Ushizawa, I. 1985. Liquid chromatographic determination of polycyclic aromatic hydrocarbons in fish and shellfish. Journal of Association of Official Analytical Chemists, 68: 945–949. Tamakawa, K. 2004. Polycyclic aromatic hydrocarbons in food. In: Handbook of Food Analysis, L. M. L. Nollet, ed. New York: Marcel Dekker, pp.1449– 1483. Tóth, L. and Blaas, W. 1972. Einfluss der Räeuchertechnologie auf den Gehalt von geraeucherten Fleischwaren an cancerogenen Kohlenwasserstoffen. II. Einfluss der Glimmtemperatur des Holzes sowie der Kuehlung, Waesche und Filtration des Räeucherrauches. Fleischwirtschaft, 52:1419– 1422. Tuominen, J., Wickström, K., Pyysalo, H. 1986. Determination of polycyclic aromatic hydrocarbons by GC-selected ion monitoring technique. Journal of High Resolution Chromatography and Chromatography Communications, 9:469–471. Vaessen, G. M. A. H., Jekel, A. A., Wilbers M. M. A. A. 1988. Dietary intake of polycyclic aromatic hydrocar-

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bons. Toxicological and Environmental Chemistry, 16:281–294. Vassilaros, D. L., Stoker, P. W., Booth, G. M., Lee, M. L. 1982. Capillary gas chromatographic determination of polycyclic aromatic compounds in vertebrate fish tissue. Analytical Chemistry, 54:106–112. Wang, G., Lee, A. S., Lewis, M., Kamath, B., Archer, R. K. 1999. Accelerated solvent extraction and gas chromatography/mass spectrometry for determination of polycyclic aromatic hydrocarbons in smoked food samples. Journal of Agricultural and Food Chemistry, 47:1062–1066. Weisshaar, R. 2002. Rapid determination of heavy polycyclic aromatic hydrocarbons in edible fats and oils. European Journal of Lipid Science and Technology, 104:282–285. White, R. H., Howard, J. W., Barnes, C. J. 1971. Determination of polycyclic aromatic hydrocarbons in liquid smoke flavours. Journal of Agricultural and Food Chemistry, 19:143–146. WHO (World Health Organization). 1987. Report of the Joint FAO/WHO Expert Commission on Food Additives. Technical Report Series, No. 31:759. Williams, P. T., Andrews, G. E., Bartle, K. D., Bishop, P., Watkins, P. 1988. Analysis of the polycyclic aromatic compounds of diesel fuel by gas chromatography with ion trap detection. Biomedical and Environmental Mass Spectrometry, 15:517–519. Wise, A. S., Sander, L. C., May, W. E. 1993. Determination of polycyclic aromatic hydrocarbons by liquid chromatography. Journal of Chromatography, 642:329–333. Yabiku, H. Y., Martins, M. S., Takahashi, M. Y. 1993. Levels of benzo(a)pyrene and other polycyclic aromatic hydrocarbons in liquid smoke flavour and some smoked foods. Food Additives and Contaminants, 10:399–405. Yang, Y., Dong, X., Jin, M., Ren Q. 2008. Rapid determination of polycyclic aromatic hydrocarbons in natural tocopherols by high-performance liquid chromatography with fluorescence detection. Food Chemistry, 110:226–232.

Chapter 20 Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene Leo M.L. Nollet

In this chapter different endocrine-disrupting compounds are discussed. The reader is directed to the other chapters to find ample information about sample preparation and cleanup techniques (for example, Figure 15.2, Bisphenol A). In Chapter 18, Personal Care Products, some further facts are presented regarding BHA (butylated hydroxyanisole) and BP-3 (benzophenone-3).

Pentachlorophenol Pentachlorophenol (PCP) (Figure 20.1) can be found in two forms, PCP or as the sodium salt. In the past, PCP has been used as a herbicide, insecticide, fungicide, algicide, disinfectant, and most of all, as an ingredient in antifouling paint. The Environmental Protection Agency (EPA) has found pentachlorophenol to potentially cause damage to the central nervous system when people are exposed to it at levels above the MCL (maximum contaminant level, 1 ppb) for relatively short periods of time. A lifetime exposure at levels above the MCL may cause reproductive effects, damage to the liver and kidneys, and cancer (Dorsey and Tchounwou 2004). The maximum contaminant level goal (MCLG) set by the EPA is zero. The EPA has set a limit for drinking water of 1 ppb. The Occupational Safety and Health AdministraAnalysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

tion (OSHA) has set a limit of 0.5 mg/m3 of workplace air for 8-hour shifts and 40-hour work weeks. Pentachlorophenol is regulated by different European Commission (EC) directives: 76/464/EEC and 2000/60/EEC, 76/769/EEC (modified by 91/173/EEC and 1999/51/EEC), 96/61/EEC, and 86/280/EEC. Regulation of pentachlorophenol is now embedded in REACH (Registration, Evaluation, Authorization and Restriction of Chemicals), COM/2003/0644 final. Environmental exposure to PCP may trigger endocrine-disrupting activities in aquatic organisms, including fish (Dorsey and Tchounwou 2004). Orton and others (2009) found in vitro and in vivo evidence of endocrine-disrupting effects of herbicides and PCP on humans and wildlife at environmentally relevant concentrations. Extraction of 2,4,6-tribromophenol (TBP), pentachlorophenol, and pentachloroanisole (PCA) from whole-fat cow milk using headspace solid-phase microextraction (HSSPME) with polyacrylate (PA) and polydimethylsiloxane (PDMS) fibers was evaluated by Mardones and others (2008). The PA fiber was studied to extract PCA, TBP, and PCP, and the PDMS fiber was used to extract PCA and acetyl derivatives of PCP and TBP. Parameters such as fiber position, matrix effect, temperature, and extraction time were studied. The analysis was made by gas chromatography (HP-5 MS column of 30 mm × 0.25 mm I.D., 0.25 μm film thickness) coupled with mass spectrometry. The 447

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Figure 20.1. Pentachlorophenol.

results showed that the distance between fiber and milk had a significant effect on the extraction kinetics. Furthermore, the fat content in milk was a critical parameter that clearly affected the extracted mass of each compound. The recoveries from milk were 95% ± 4%, 96% ± 2%, and 96% ± 4% for PCP, TBP, and PCA, respectively, with the PDMS fiber and 94% ± 3% and 95% ± 1% for TBP and PCA, respectively, with PA fiber. Solid-phase extraction joined with dispersive liquid–liquid microextraction (DLLME) has been developed as an ultrapreconcentration technique for the determination of chlorophenols in water samples (Fattahi and others 2007). Chlorophenols (CPs), such as 2-chlorophenol (2-CP), 3-chlorophenol (3-CP), 4-chlorophenol (4CP), 2,6-dichlorophenol (2,6-DCP), 2,5-dichlorophenol (2,5-DCP), 2,4-dichlorophenol (2,4-DCP), 3,5-dichlorophenol (3,5-DCP), 2,3-dichlorophenol (2,3-DCP), 3,4-dichlorophenol (3,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), 2,3,6-trichlorophenol (2,3,6TCP), 2,3,5-trichlorophenol (2,3,5-TCP), 2,4,5-trichlorophenol (2,4,5-TCP), 2,3,4-trichlorophenol (2,3,4-TCP), 3,4,5-trichlorophenol (3,4,5-TCP), 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP), 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP), 2,3,4,5-tetrachlorophenol (2, 3,4,5-TeCP), and pentachlorophenol were determined by gas chromatography–electron capture detection (BPX5 capillary column, 30 m × 0.25 mm I.D., 0.25 μm film thickness). DLLME is a miniaturized sample pretreatment technique that uses microliter volumes of the extraction solvent. Simplicity of operation, rapidity, low sample volume,

low cost, and high enrichment factor are some advantages of DLLME. In the article by David and Sandra (2007), an overview is given of stir bar sorptive extraction for trace analysis of, for example, pentachlorophenol. A robustness test of a solid-phase microextraction-based method optimized for the simultaneous determination of chloroanisoles and acetyl-chlorophenols implicated in the presence of corky taste in wine has been performed by Pizarro and others (2008). Chromatographic analyses were performed with a gas chromatograph equipped with a split/splitless injector, electronic pressure control in the injector, and ECD detection. A capillary column HP-5MS (30 m × 0.25 mm I.D., 0.25 μm film thickness) was used. A headspace solid-phase microextraction (HS-SPME) method was optimized for the analysis of three chloroanisoles (2,4,6-trichlorochloroanisole, 2,3,4,6-tetrachlorochloroanisole, and pentachloroanisole), as well as their precursor chlorophenols (2,4,6-trichloro chlorophenols, 2,3,4,6-tetrachloro chlorophenols, and pentachlorophenol) involved in the presence of cork taint in wine. The origin of these chloroanisoles, which are directly responsible for this off-flavor, is related to the microbial degradation of the corresponding chlorophenols used as insecticides in different wood materials present in the wineries and the microflora and hypochlorite used to bleach wine corks. Lambropoulou and others (2007) discuss recent developments in headspace microextraction techniques for the analysis of environmental contaminants. Llompart and others (2002) developed an HS-SPME/GC–MS method for the determination of lowconcentration phenolics and halogenated phenolics in water. Two of the five coatings, CAR-PDMS 85 μm and PDMS 100 μm were the most suitable fibers and have been used for the determination of 30 phenolics in water. The first one is especially appropriate for phenol, methylphenols, and low chlori-

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

nated chlorophenols, and the second one for highly chlorinated phenols. No matrix effect was observed for most of the analytes, with the exception of tetrachlorophenols and pentachlorophenol. Basheer and Lee (2004) have compared HF-LPME (hollow-fiber liquid-phase microextraction) and HS-SPME for the determination of alkylphenols (APs), chlorophenols (CPs), and bisphenol-A (BPA) in aqueous samples. The HS-SPME procedure involved on-fiber derivatization by exposing the fiber to the headspace of a derivatizing solution (BSTFA). HS-SPME gave a precision of between 5.9% and 13.9% (with 60-min extraction time), which is comparable with that obtained by the proposed LPME method (RSDs between 5.2% and 17.7%), but the extraction time was only 30 min. The results showed that LPME–GC injection portderivatization is a promising method for APs, CPs, and BPA and has good agreement with the performance of SPME. Polo and others (2006) have developed an in situ acetylation/HS-SPME/GC–MS method for the simultaneous determination of brominated phenols and other phenolic pollutants in complex water samples. Zhou and others (2005) developed an HS-SPMEGC method for the determination of the phenolic compounds in water samples. Martínez-Uruñuela and others (2004) developed a method based on HS-SPME for the determination of acetylated chlorophenols in wine samples. According to this study, HS-SPME proved to be an adequate and potential alternative to the traditional technique based on LLE for the direct determination of chlorophenols in wine samples. This method is free of organic solvents, simpler, and faster than that used so far by achieving LODs < 0.020 μg/L with a GC–ECD system. Insa and others (2004) have also used a HS-SPME method coupled with in situ derivatization for the determination of chlorophenolic compounds in cork-soaking solu-

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tions (ethanol/water mixtures, 12% v/v ethanol) or in wine samples. Very recently, an interesting report on phenolic compounds analysis came from Hernández-Córdoba’s group (Campillo and others 2006). In this study, HS-SPME was combined with GC-AED (atomic emission detector) (30 m × 0.32 mm I.D. HP-5, 5% diphenyl, 95% dimethyl polysiloxane capillary column) for the determination of nine phenolic compounds in honey samples. The PDMS/DVB 65 μm fiber was the most suitable for preconcentrating the analytes from the headspace of an aqueous solution containing the dissolved honey samples where the chlorophenols had been submitted to acetylation. The optimized method was successfully applied to different samples; some of the studied chlorophenols being detected in some of the analyzed honey at concentration levels of 0.6–9.4 ng/g. MHS-SPME (multilple headspace-SPME) coupled to GC-MS/MS has been evaluated and presented as an alternative to HS-SPME by Martínez-Uruñuela and others (2005) for the quantitative analysis of chlorophenols and chloroanisoles related to cork taint in wine. The gas chromatograph was connected to an ion-trap mass spectrometer. Compounds were separated using a VF-5 MS capillary column (30 m × 0.25 mm I.D., 0.25 μm film thickness). Pizarro and others (2007a, b) presented a method based on the use of microwaveassisted extraction (MAE) for the quantitative analysis of 2,4,6-trichloroanisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA), pentachloroanisole (PCA), 2,4,6-tribromoanisole (TBA), 2,4,6-trichlorophenol (TCP), 2,3,4,6tetrachlorophenol (TeCP), pentachlorophenol, and 2,4,6-tribromophenol (TBP) in cork stoppers. The chromatographic analysis was performed with a gas chromatograph equipped with a splitless injector, electronic pressure control in the injector, and an electron-capture detector. The column used was a capillary column HP-5MS (30 m × 0.25 mm I.D., 0.25 μm film thickness).

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A sensitive method based on pressurized liquid extraction (PLE) and liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) has been developed by Carabias-Martínez and others (2006) to determine seven endocrine-disrupting compounds in cereal samples: bisphenol A (BPA), 4-tert-butylbenzoic acid (BBA), 4-nonylphenol (NP), 4-tert-butylphenol (t-BP), 2,4dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP), and pentachlorophenol. LC–MS analysis was performed using an Alliance HT 2795 HPLC system connected to a ZQ 4000 quadrupole mass spectrometer system with electrospray ionization (ESI) interface. The chromatographic column was a 2.1 mm × 100 mm XTerra MS C18 with 3.5-μ particle size. A simultaneous determination of the chloroanisoles and chlorophenols in cork samples was performed with GC-ECD (Insa and others 2004, 2006). A DB-5 capillary column (30 m × 0.25 mm I.D., film thickness 0.25 μm) was used. A sensitive analytical method based on liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) has been developed for the determination of seven endocrine-disrupting compounds: 4n-nonylphenol (NP), 4-tert-butylphenol (tBP), bisphenol A (BPA), 2,4-dichlorophenol (DCP), 2,4,5-trichlorophenol (TCP), PCP and 4-tert-butylbenzoic acid (BBA) in water samples by Carabias-Martínez and others (2004). Analytes were solid-phase extracted. The chromatographic column was a 100 m × 2.1 mm XTerra MS C18 with 3.5 μm particle size. The quadrupole mass spectrometer system could use either APCI or ESI interfaces. Postcolumn addition of a volatile strong base, 1,8-diazabicyclo-(5,4,0) undec-7-en (DBU), was performed to enhance sensitivity by ESI-(NI)-MS detection. A method for the simultaneous measurement of trace amounts of phenolic xenoestrogens, such as 2,4-dichlorophenol (2,4-DCP), 4-tert-butylphenol (BP), 4-tert-octylphenol

(OP), 4-nonylphenol (NP), pentachlorophenol (PCP), and bisphenol A (BPA) in water samples was developed using stir bar sorptive extraction (SBSE) with in situ derivatization followed by thermal desorption (TD)–gas chromatography–mass spectrometry (GC– MS) analysis (Kawaguchi and others 2004). The injection was performed in the splitless mode. The separation was accomplished on a DB-5MS fused silica column (30 m × 0.25 mm I.D., 0.25 μm film thickness). The mass spectrometer was operated in the selected ionmonitoring (SIM) mode with electron impact ionization. After stir bar sorptive extraction, 2,4,6-trichloroanisole, 2,3,4,5-tetrachloroanisole, pentachloroanisole, and their respective phenols from synthetic and real wine samples were analyzed by GC-MS (Zalacain and others 2004). The column was a fused silica capillary column (BP21 stationary phase 50 m length, 0.22 mm I.D., and 0.25 μm film thickness). For mass spectrometry analysis, electron impact mode was used. Considering their use in plastic materials intended to come into contact with foodstuffs, five of the compounds studied—BBA (4-tertbutylbenzoic acid), NP (4-nonylphenol), PCP, BPA (bisphenol A), and t-BP (4-tertbutylphenol)—have been examined or are currently being evaluated by the Scientific Committee for Food (SCF) of the European Commission in order to develop future legislation concerning these chemicals (European Commission 2003). Thus, several authors have focused their investigations on the determination of these compounds. For example, in the analysis of virgin/recycled paper products in contact with food, Ozaki and others (2004) found some EDCs, among which BPA and PCP were included. Finally, some older methods are discussed. Cooper and others (1994) worked out an analysis method of pentachlorophenol residues in wine corks. This method was based on GC connected to an ELCD (electrolytic conductivity detector) (megabore column,

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

30 m length, 0.53 mm I.D., 1.5 μm film thickness) and/or MS (capillary column DB 17, 30 m, 0.32 mm I.D., 0.25 μm film thickness). Pentochlorophenol and neutral organochlorine compounds, such as hexachlorobenzene or DDT, were quantified by GC-ECD in human milk (Butte and Fooken 1990). GC was performed on a SE 54 fused silica capillary column (50 m × 0.2 mm) connected with a 63Ni electron capture detector. Concentrations of PCP in human milk were found to be 0.039 mg/kg (fat basis). Chlorinated phenols ranged in human milk from 0.75 to 9.74 × μg kg−1 (Veningerova and others 1996). The determination limit of PCP found was 1.0 μg kg−1. Compounds were derivatized with pentafluorobenzyl bromide, analyzed on a PTE-5 GC column (30 m length, 0.25 mm I.D., 0.25 μm film thickness), and quantified by ECD.

Benzophenone Benzophenone sunscreen compounds are used as ultraviolet absorbers and fragrance

451

retention agents in the manufacture of cosmetics and pharmaceuticals (Table 20.1). Benzophenones (BPs) demonstrate maximum absorption at wavelengths of 288–290 and 325 nm, and absorb light in the 200- to 400-nm wavelength region (Rieger 1997). Consequently, BPs may absorb ultraviolet light harmful to the human body: UVA (320– 400 nm) and UVB (290–320 nm). They may be effective in preventing skin disorders and skin cancer (Moloney and others 2002). A number of studies, however, have revealed estrogenic activity of BPs (Miller and others 2001; Schlumpf and others 2001; Takatori and others 2003). Furthermore, BPs may have an impact on the ecosystem (Giokas and others 2007). The BP 2-hydroxy-4-methoxybenzophenone (oxybenzophenone, BZ-3 or BP-3) has the ability to absorb ultraviolet light. Therefore, it is used in many cosmetics and sunscreens to protect human skin from ultraviolet radiation. It is used as a photostabilizer for agricultural films and paints. The U.S. Food and Drug Administration (FDA) has

Table 20.1. Benzophenones and related products. Compound Benzophenone Benzophenone-8 Benzophenone-9 Benzophenone-1 Benzophenone-2 Benzophenone-3 Benzophenone-4 Benzophenone-5 Benzophenone-6 Benzophenone-7 Benzophenone-10 Benzophenone-11 Benzophenone-12

4-Methylbenzophenone

Chemical Nature 2,2′-Didydroxy-4-methoxybenzophenonedioxybenzone 2,2′-Dihydroxy-4,4′-dimethoxybenzophenone5,5′-disulfonic acid sodium salt 2 ,4-Dihydroxybenzophenone 2,2′,4,4′-Tetrahydroxybenzophenone 2-Hydroxy-4-methoxybenzophenone-oxybenzone 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid-sulisobenzone 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid, monosodium salt 2,2′-Dihydroxy-4,4′-dimethoxybenzophenone 5-Chloro-2-hydroxybenzophenone 2-Hydroxy-4-methoxy-4′-methylbenzophenonemexenone Bis(2,4-ihydroxyphenyl)methanone 2-Hydroxy-4(octyloxy) benzophenone-octabenzone 2,3,4-trihydroxybenzophone 4-hydroxybenzophenone Benzhydrol (4-Methylphenyl)phenylmethanone

Abbreviation BP

CAS No. 119-61-9 131-53-3 76656-36-5

DHB HMB–BZ-3

131-56-6 131-55-5 131-57-7 4065-45-6 6628-37-1

DHMB

131-54-4 85-19-8 1641-17-4 1341-54-4 1843-05-6

THB HBP BH 4-MBP

134-84-9

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Analysis of Endocrine Disrupting Compounds in Food

also approved benzophenone-3 for use as an indirect food additive (Okereke and others 1995). Benzophenone-3 absorbs from 200 to 400 nm, with maximum absorbances at 288–290 and 325 nm (λmax) (Rieger 1997). A few of the metabolites of benzophenone-3, such as 2,4-dihydroxybenzophenone and 2,2 ′ - dihydroxy - 4 - methoxybenzophenone, are also used in sunscreens (Okereke and others 1993). BZ-3 may be absorbed through the skin and accumulate in the body. It is passed through the kidneys, metabolized, and excreted in the urine. Benzophenone-3 is metabolized into at least three metabolites: 2,4-dihydroxybenzophenone (DHB), 2,2′dihydroxy-4-methoxybenzophenone (DHMB), and 2,3,4-trihydroxybenzophenone (THB) (Okereke and others 1993). Sample preparation methods, such as online continuous liquid–liquid extraction (LLE) (Soliman and others 2004) and solidphase extraction (SPE) (Castillo and others 1997; Giokas and others 2004), have been developed for the determination of BPs. However, LLE requires large volumes of organic solvents and a number of concentration steps. Recently, solid-phase microextraction (SPME) was used for the determination of BPs in water samples (Salafranca and others 2003). However, the limit of detection (LOD) of BP in a river water sample was 4.2 μg/L−1, and its sensitivity was low. UV curing inks contain starting molecules for the polymerization (Eurofins, 2009). In the rapid alert system for food and feed of the European Union (RASFF), from February 2009, the detection of two substances was reported. Benzophenone and 4-methylbenzophenone were found in cereal products from Belgium in concentrations up to 4 mg/kg. The tolerable daily intake of benzophenone is 0.01 mg/kg BW. A specific migration limit of 0.6 mg/kg food is fixed in the plastic directive 2002/72/EC. For 4-methylbenzophenone, no toxicological data are available as of 2010.

The European Food Safety Authority (EFSA) was asked to provide a risk assessment. The Panel on Food Contact Materials (EFSA 2009) considered the EFSA approach to derive a lowest observed adverse effect level (LOAEL) of 6 mg/kg BW benzophenone per day as conservative. The panel derived a tolerable daily intake (TDI) of 0.03 mg/kg BW per day. This value is proposed as a basis for calculation of the margin of exposure (MoE) for 4-methylbenzophenone. A multiresidue method was elaborated by R. Rodil and others (2009) for BP-3, BP-4, and a great number of other emerging pollutants in tap water, surface water, and wastewater. After SPE, compounds were detected by LC in combination with triple quadrupole MS with electrospray ion source. With an automated on-line columnswitching HPLC-MS/MS method with peak focusing, BP-3, parabens, triclosan, and environmental phenols were detected in human milk (Ye and others 2008). The column of SPE was Lichrosphere RP-18ADS (25 mm × 4 mm, 25 μm particle size, 60 Å pore size); the HPLC column was Zorbax Eclipse XDD-C8 (150 mm × 4.6 mm, 5 μm). A trap mass spectrometer with atmospheric pressure photoionization (APPI) interface was used. The SPE recovery of BP-3 was 105%; the LOD was 0.4 mg/mL. Hee-Kyung Jeon and others (2006) developed a method to simultaneously determine seven UV filters in, for example, surface water and soils. The UV filters determined were benzophenol, benzhydrol (BH), 4hydroxybenzophenone (HBP), 2-hydroxy4-methoxybenzophenone (HMB), 2,4-dihydroxybenzophenone (DHB), 2,2′-dihydroxy4-methoxybenzophenone (DHMB), and 2,3,4-trihydroxylbenzophenone. UV filters in water samples were extracted by liquid–liquid partitioning. The analytes were derivatized with N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA). Determination was by GC-MSD. The

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

column used was an Ultra 2 capillary column of 30 m length and 200 μm I.D., with 0.33 μm film thickness. Detection limits ranged from 5 to 100 ng/L. A technique was developed to determine benzophenone-3 and its metabolites in water and urine (Felix and others 1998). Extraction was by SPME and detection by GCquadrupole ion trap MS. The column used was a DB5-MS column. Guillot and others (2006) compared the official EU liquid–liquid extraction (LLE) method with solid-phase microextraction (SPME) for the analysis of compounds migrating from cross-linked polyethylene, such as benzophenone, into water. A medium polarity polydimethylsiloxane/divinylbenzene (PDMS/DVB) 65 μm fiber proved most efficient for the SPME extraction of benzophenone and the other eight test compounds. Four preservatives (MP, EP, BP, and PB), two antimicrobial agents (triclocarban and triclosan), and five UV filters (benzophenone-1, benzophenone-3, benzophenone-8, octocrylene, and octyldimethylp-aminobenzoic acid) were determined in surface waters and wastewaters by ultra-highperformance LC-tandem MS (Pedrouzo 2009). Benzophenone-3 had the highest concentration levels (7 ng/L) in river waters. Negreira and others (2009) developed a sensitive determination method for salicylate and benzophenones (benzophenone-1, benzophenone-3, and benzophenone-8) in water samples using SPME, derivatization, GC, and MS. A PDMS-DVB-coated fiber was used. The headspace was on fiber silylated with MSTFA. Separation was performed with an HP-5MS capillary column (30 m × 0.25 mm I.D., 0.25 μm). Gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) techniques were developed for the determination of five ink photoinitiator residues in packaged food beverages: 2-isopropylthioxanthone (ITX),

453

benzophenone, 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB), 1-hydroxycyclohexyl1-phenyl ketone (IRGACURE 184), and ethyl-4-dimethylaminobenzoate (EDAB) (Sagratini and others 2008). Samples were extracted from milk, fruit juices, and wine, and their respective packaging, using nhexane and dichloromethane. After purification on SPE silica gel cartridges, samples were analyzed with GC-MS (HP 5 MSI column, 30 m × 0.25 mm, 0.25 μm film thickness) and LC-MS (Luna C18, 250 mm × 4.6 mm I.D., 5 μm). The recovery percentages, obtained by spiking the beverage samples at concentrations of 4 and 10 μg/ L−1 with a standard mixture of photoinitiators, were in the range 42%–108% (milk), 50%– 84% (wine), and 48%–109% (fruit juices). The LODs and LOQs, obtained using GCMS, were in the range 0.2–1 and 1–5 μg/L−1, respectively. The method was applied to the analysis of 40 packaged food beverages (milk, fruit juices, and wine samples). The most significant contamination was that of benzophenone, found in all samples in a concentration range of 5–217 μg/L−1. Its presence was confirmed by an LC/APPI/MS/MS analysis.

Parabens Parabens are a group of chemicals widely used as preservatives in the cosmetic and pharmaceutical industries. The commercially known parabens (Núñez and others 2008) are used as preservatives and bactericides in personal care products, pharmaceutical preparations, and food and beverages (Labat and others 2000; Lokhnauth and others 2005; Saad and others 2005) due to their stability and low volatility. Parabens are esters of para-hydroxybenzoic acid. Common parabens include methylparaben (MePor MP; E number E218), ethylparaben (EtP or EP; E214), propylparaben (PrP or

454

Analysis of Endocrine Disrupting Compounds in Food

Figure 20.2. Chemical structure of paraben.

PP; E216), and butylparaben (BuP or BP). Less common parabens include isobutylparaben, isopropylparaben, benzylparaben, and their sodium salts. The general chemical structure of a paraben (R = alkyl group) is shown in Figure 20.2. Parabens are rapidly absorbed, metabolized, and excreted. Major metabolites of parabens are p-hydroxybenzoic acid (pHBA), p-hydroxyhippuric acid, p-hydroxybenzoyl glucuronide, and p-carboxyphenylsulfate. Studies, in vivo and in vitro, have reported the estrogenic activity of parabens (Silva and others 2002; Darbre and others 2004; Golden and others 2005; Soni and others 2005; Rudel and others 2003). This activity depends on the length and ramification of the chain. Parabens with shorter side chains are less potent estrogens than those with longer or branched side chains (Routledge and others 1998; Golden and others 2005). It has been demonstrated that parabens have not only estrogen agonist properties but also androgen antagonist activity (Darbre and others 2008). MeP and PrP are the most commonly employed parabens, and they are often used together because they have synergistic effects. In recent years, some researches reported cases of dermatitis and the presence of parabens in tissue samples from human breast tumors (Darbre and others 2004). Parabens appear at high concentrations in personal care products; the concentrations of individual compounds in creams are in the range of 114–1044 μg/g−1 (Zhang and others 2005). The European Directive 76/768/EC allows their use in such products with a

maximum authorized concentration of 0.4% (w/w) for a single ester and 0.8% (w/w) for ester mixtures, expressed as p-hydroxybenzoic acid (The Council Directive 76/768/EC, 1976). The EFSA concluded in September 2004 that a group acceptable daily intake (ADI) of 0–10 mg/kg body weight per day could be established for MP and EP and their sodium salts but not for PP (European Commission 2005). For quantification of phenols such as triclosan or parabens in milk, gas chromatography–mass spectrometry was used (GC–MS) (Adolfsson-Erici and others 2002; Dayan 2007; Allmyr and others 2006a, b). Triclosan (2,4,4′-trichloro-2′-hydroxydiphenyl ether, or TCS) is a synthetic, broad-spectrum antibacterial agent used in toothpaste, mouthwash, and other items. However, GC methods usually require a relatively large amount of sample (3–10 mL), laborious sample cleanup (e.g., liquid–liquid extraction), and sometimes a derivatization step because of the relatively low volatility of the compounds. Ye and others (2008) developed a sensitive method using an on-line solidphase extraction high performance liquid chromatography-tandem mass spectrometry system with a peak focusing feature to measure the concentrations of five parabens (methyl-, ethyl-, propyl-, butyl-, and benzyl parabens), triclosan, and six other environmental phenols: bisphenol A (BPA); orthophenylphenol (OPP); 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; and 2-hydroxy-4-methoxybenzophenone (BP-3) in human milk (Ye and others 2008). The method, validated by use of breast milk pooled samples, showed good reproducibility and accuracy. The detection limits for most of the analytes were below 1 ng mL−1 in 100 μL of milk. The method was tested by measuring the concentrations of these 12 compounds in four human milk samples. Methyl paraben, propyl

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

paraben, triclosan, BPA, OPP, and BP-3 were found in some of the samples. The free species of these compounds appear to be the most prevalent in milk. Alkylparabens were separated by μHTLC-ELSD (micro-high-temperature liquid chromatography-evaporative light-scattering detector) in pharmaceutical analysis (Guillarme and others 2008). Canosa and others (2007) evaluated the suitability of PLE (pressurized liquid extraction) for the simultaneous extraction and purification of TCS and four alkyl parabens (methyl, ethyl, propyl, and butyl) from dust samples. After extraction, analytes were derivatized and determined by GC– MS/MS, using the conditions of Canosa and others (2007). This is important because humans, particularly infants, are exposed to dust through inhalation and oral ingestion. In the study by Xiu Qin Li and others (2008), HPLC/TOF-MS (time-of-flight-MS) was used to detect and quantify 18 preservatives in beverages. Preservatives analyzed were MP, EP, PP, isopropylparaben (IPP), BP, isobutylparaben (IBP), hexylparben (HP), 2-naphthol (2-NL), 2-phenylphenol (OPP), 4-hexylresorcinol (4HR), thiabendazole (TBZ), imazalil (IMZ), 2,4dichlorophneoxyacetic acid (2,4-DA), benzyldodecyldimethylammonium bromide (BDDAB), and nordihydroguaiaretic acid (NDGA). Test samples included carbonated fruit and fruit juice beverages from local supermarkets in China. Sample preparation consisted of degassing and SPE. The HPLC column was a reversed-phase C18 column (150 mm × 2.1 mm, 3.5 μm particle size, Zorbax Eclipse XDB-C18). Mobile phase B was acetonitrile, and mobile phase A was water (negative ion mode) and 20 mM ammonium acetate, with 0.1% formic acid (positive ion mode). Limits of detection ranged from 0.0005 to 0.05 mg/kg, far below the required maximum residue levels.

455

García-Jiménez and others (2007) discussed the analysis of a number of additives used in foods and cosmetics with different polarities, namely aspartame (AS), acesulfame (AK), saccharin (SA), MP, EP, PP, BP, propyl gallate (PG), and butylated hydroxyanysole (BHA). The monolithic column used as the separation system was a 5-mm commercial precolumn of silica C18 coupled to a flow injection manifold working with a peristaltic pump. The mixture was separated in only 400 s. To achieve the separation in the FIA system two carriers were used: first, a mixture of acetonitrile-water buffered with 10 mM pH 6.0 phosphate buffer, and second, a methanol-water mixture to improve the carrier strength and speed up the more apolar analytes at 3.5 mL/min−1. Detection was by means of a diode array spectrometer (DAD) at the respective wavelength of each compound. A Hewlett-Packard 1100 series liquid chromatograph with a DAD detector provided with a C8 Zorbax column was used to validate the method. A total of 67 food samples were purchased from supermarkets located in the northern states (Kedah and Perlis) of Peninsular Malaysia. The samples were categorized as soft drinks (9), canned fruits/vegetables (19), jam/fruit jelly (11), sauces (15), dried fruits (8), and miscellaneous (5) (Saad and others 2005). A reversed-phased HPLC method that allows the separation and simultaneous determination of the preservatives benzoic (BA) and sorbic acids (SA), MP, and PP was described. The separations were effected by using an initial mobile phase of methanol– acetate buffer (pH 4.4) (35 : 65) to elute BA, SA, and MP and changing the mobile phase composition to methanol–acetate buffer (pH 4.4) (50 : 50) thereafter. Detection wavelength was 254 nm. Analytical separation was carried out using a Supelco 516 C18 column (15 cm × 4.6 mm, 5 μm) at room temperature. The developed method was applied to the determination of 67 foodstuffs. The range of

456

Analysis of Endocrine Disrupting Compounds in Food

preservatives found were from not detected (nd)–1260, nd–1390, nd–44.8, and nd– 221 mg/kg−1 for BA, SA, MP, and PP, respectively. A method for the simultaneous determination of parabens, including MP, EP, PP, and BP by HPLC (Zorbax Eclipse XDBC8, 150 × 4.6 mm I.D., 5 μm) coupled with chemiluminescence detection, was developed by Zhang and others (2005). The procedure was based on the chemiluminescent enhancement by parabens of the cerium(IV)–rhodamine 6G system in a strong sulfuric acid medium. The good separation of parabens was carried out with an isocratic elution using a mixture of methanol and water (60 : 40, v/v) within 8.5 min. Under the optimized conditions, the detection limits were 1.9 × 10−9, 2.7 × 10−9, 3.9 × 10−9, and 5.3 × 10−9 g mL−1 for MP, EP, PP, and BP, respectively. The chemiluminescence reaction was quite compatible with the mobile phase of HPLC. The proposed method has been successfully applied to the assay of parabens in wash-off cosmetic products and foods with minimal sample preparation. All of the food samples were obtained at local markets. Liquid samples including orange juice, soy sauce, vinegar, and cola soda were prepared by diluting 1 g of sample with 50 mL of mobile phase. Pickled Chinese artichoke was cut up and prepared by blending 10 g of the sample with 50 mL of mobile phase and sonicating for 10 min. The blended sample was then allowed to settle for 10 min, and 1 mL of the supernatant liquid was diluted 1 : 10 with mobile phase. Strawberry jam was prepared just like the pickle sample, except for cutting. After dilution, all sample solutions were filtered through a 0.22 μm Millipore membrane to remove particulate matter from the samples. The determination of a group of parabens including MP, EP, PP, and BP by using the flow injection analysis technique with chemiluminescence (CL) detection has been carried out (Myint and others 2004). The

method is based on the enhancement by parabens of cerium(IV)-rhodamine 6G chemiluminescence reaction in sulfuric acid medium. The method shows higher sensitivity than most of the reported methods, which also require preconcentration or derivatization. It was successfully applied to the determination of ethylparaben in soy sauces without tedious pretreatment. Separation was by gas chromatography. The capillary column was a CP Wax 52 CB, 30 m × 0.25 mm I.D. with 0.25 μm film thickness. Lee and others (2006) evaluated supercritical fluid extraction (SFE) in combination with LC-MS to determine not only MP, EP, PP, and BP but also butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, and α-tocopherol acetate in cosmetic products. SFE has the following advantages: less consumption of hazardous organic solvents, reduction in laboratory labor, and higher sample throughput. Chromatographic separation was performed with a C18 column (5 μm) 4.6 mm × 250 mm. Gradient elution was with 100% methanol (solvent A) in 100% water (solvent B). Detection limits for MP, EP, PP, and BP were 4.7, 13.5, 13.4, and 19.3 ng/g, respectively. SPE in combination with thermal desorption-GC (TD-GC) was applied for the determination of MP, EP, PP, benzoic acid, and sorbic acid in soft drinks, yogurts, and sauces (Wang and others 2006). Instrumentation was a double-shot pyrolyzer attached to a gas chromatograph. Detection was by flame ionization (FID). The column used was a capillary Ultra Alloy-17 column (30 m × 0.25 mm I.D.). The limit of detection ranged from 0.002 to 0.2 mg/L. An in situ derivatization SPME method was developed for the analysis of parabens, triclosan, and related chlorphenols in water (Regueiro and others 2009). On-fiber silylation used N-methyl-N-(tert-butyldi-

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

methylsilyl)trifluoroacetamide (MTBSTFA). Separation was carried out on an HP-5MS capillary column (30 m × 0.25 mm I.D., 0.25 μm film thickness). The ion trap mass spectrometer was operated in the electron impact ionization-positive mode. The concentrations from different river waters of MP, EP, PP, and BP ranged from not quantified to not detected, from not quantified to not detected, from not quantified to 23.8 pg mL−1, and from not detected to 54.1 pg mL−1, respectively for different river waters. Blanco and others (2009) developed a method for the determination of MP, EP, PP, BuP, and BeP in environmental water, such as tap water. After off-line SPE extracts were analyzed by nonaqueous capillary electrophoresis (NACE) coupled with DAD, large-volume sample stacking (LVSS) based on the electroosmotic flow pump was used. Concentrations found in tap water were 0.04 ng mL−1 for MP, were not detectable for BuP and EP, and were lower than the LOQ for PP. In Kulikov and Verushkin (2008) the simultaneous determination of paracetamol, caffeine, guaifenesin, benzoic acid, MP, and PP in syrups is discussed. The LC separation used a Kromasil C18 column (150 mm × 4.6 mm, 5 μm) and a mobile phase of 1-butanol-water (1 : 99 v/v), containing 0.04 M sodium dodecyl sulfate and 0.1% (v/v) trichloroacetic acid. Detection was at 260 nm. MP, EP, PP, isopropylparaben, BuP, isobutylparaben, and heptylparaben, were analyzed in foodstuff by Xiu-Qin and others (2008). An Acquity C18 column (50 × 2.1 mm I.D., 1.7 μm particle size) was used. The mobile phase was a mixture of acetonitrile and 20 mM potassium dihydrogen phosphate/ phosphoric acid (pH 4.29) buffer. Detection was by DAD. Analysis of various p-hydroxybenzoic esters (methyl, ethyl, isopropyl, propyl, benzyl, and butylparaben) in environmental solid samples was carried out by sonication-

457

assisted extraction in small columns (SAESC) followed by liquid chromatography with triple quadrupole mass spectrometry (MS/ MS) (Nunez and others 2008). Solid samples were placed in small glass columns and extraction was performed with the assistance of sonication in two consecutive steps of 15 min using acetonitrile as extraction solvent. Sample extracts were evaporated under a nitrogen stream to 1 mL and analyzed by LC–MS/MS. After sonication-assisted extraction, satisfactory recoveries were obtained ranging from 83% to 110%, depending on the analyte. Good limits of quantification (LOQs), between 0.11 and 0.49 ng g−1 were obtained for LC–MS/MS, making this technique suitable for the determination of parabens in environmental solid samples, particularly at trace levels. For further examples see Table 20.2.

Butylated hydroxyanisole Butylated hydroxyanisole (BHA, E320) (Figure 20.3) is used as an antioxidant and food preservative in food packaging, cosmetics, pharmaceuticals, and animal feed. BHA has an ADI of 0–0.5 mg/kg BW. The use of synthetic antioxidants is regulated by legislation in most countries, the limit amounts that can be added to foods being strictly defined. In the European Union, for example, the amount of synthetic antioxidants is limited to 0.01% (0.1 g/kg) for each antioxidant if used individually

Figure 20.3. BHA (a) 3-tert-butyl-4-methoxyfenol; (b) 2-tert-butyl-4-methoxyfenol.

458

Benzoic acid, p-hydroxybenzoic acid (PBA), MP, EP, PP

MP, EP, PP, BP

MP, EP, PP

Haloperidol, MP, EP, n propylparaben, iso propylparaben, n-butylparaben, iso-butylparaben, sec butylparaben, 4-(4-chlorophenyl)-4hydroxypiperidine, 4-fluorobenzoic acid and 4-hydroxybenzoic acid MP, EP, isopropyl, n-propyl, isobutyl, and n-butyl p-hydroxybenzoic acid esters Phenoxetol, MP, EP, n-propyl paraben, iso-butyl paraben, n-butyl paraben, and croconazole HCl

CE

CE

HPLC

CE

HPLC

HPLC

MP, PP

Analytes

HPLC

Separation Method

Mayonnaise

RP-HPLC

RP-HPLC

Isocratic

Water-methanol (50 : 50)

Derivatization with 4-bromomethyl-6,7-dimethoxycoumarin Migrating solution containing 10 mM SDS, 10 mM phosphate buffer at pH 7.0, and 15% ethanol. Electrophoretic conditions: 30 kV, 45°C capillary temperature

Running buffer, 20 mM sodium borate buffer, pH 7.8, containing 7.5 mM SDS 30 kV at 25°C

Silica capillary (50 μm I.D.m × 65 cm long, 56.5 cm to detector) Spectraphoresis 100 capillary electrophoresis system. Capillary: 75 μm I.D., 190 μm O.D., 96 cm length, and 60 cm to detector. C18 column

Liquid formula medicines Cosmetic products

Methanol and phosphate buffer, pH 2.5 (65 : 35, v/v)

Mobile Phase–Derivatization–Buffers

5 μm SUPELCO Discovery column C18 (125 mm × 4 mm I.D.)

Column

Emulgel

Products

Table 20.2. HPLC and GC methods of parabens.

FD: λex. = 355 nm λem. > 420 nm 200 nm

255 nm

PAD 205 nm

245 nm

Detection

Maeda and others 1987 Akhtar and others 1996

Burini 1994 Driouich and others 2000

Hajkova and others 2002 Heo and Lee 1998 Wang and Chang 1998

Reference

459

MP, EP, PP, BP

MP, PP, thimerosal

MP, EP, PP, BP

Benzoic acid p-hydroxybenzoic acid BP, EP, PP

HPLC/CE

HPLC

GC

HPLC

Soy sauce

Pharmaceutical products

Pharmaceutical products

Commercial cosmetic product

Cosmetic cream

Products

3% SE-30 column (200 cm × 2 mm I.D.) 3% QF-1 column (200 cm × 2 mm I.D.) 2% OV-1 column (200 cm × 4 mm I.D.) Lichrospher RP-18 (4.0 mm I.D.m × 25 cm)

C18-bonded silica column

HPLC: reversed-phase Lichrospher C18 column (125m × 4 mm I.D., 5 μm) CE: silica capillary tube (ThermoQuest) (70 cm × 75 μm I.D, 63 cm length)

Hypersil BDS cyanopropyl column (200m × 4.6 mm I.D.)

Column

Acetonitrile: 0.03 M NaH2PO4 (42 : 58, v/v)

Derivatization: hexamethyldisilazane + heptafluorobutyric anhydride Carrier gas: nitrogen

Methanol-monobasic sodium phosphate (pH 3.5; 0.025 M) (40 : 60, v/v). The composition was altered gradually to 80% of methanol over 8 min. This composition was maintained for 4 min. The initial eluent composition was restored in 5 min. Flow rate: 1.0 mL min−1. Gradient of methanol (A) and water-acetic acid (1%) (B). The gradient was as follows: 0 min: 35% A; 13 min: 60% A; 25 min: 60% A Flow rate was 1.0 mL/min Buffer :15 mM sodium tetraborate (pH 9.2) with methanol (85 : 15, v/v) 40°C 20 kV Methanol and aqueous 0.02 M phosphoric acid (59 : 41, v/v).

Mobile Phase–Derivatization–Buffers

215 nm UV-Vis

F.I.D.

E.D.

295 nm UV-Vis detector

220 and 240 nm UV-DAD

Detection

Chu and others 2003

Kang and Kim 1997 De Croo and others 1984

Labat and others 2000

Sottofattori and others 1998

Reference

HPLC, high-performance liquid chromatography; RP-HPLC, reversed-phase high-performance liquid chromatography; CE, capillary electrophoresis; GC, gas chromatography; MP, methylparaben; EP, ethylparaben; PP, propylparaben; BP butylparaben; SDS, sodium dodecyl sulphate; PAD, pulsed amperometric detection; FD, fluorometric detection; UV-DAD ultraviolet diode array spectrometer; E.D., electrochemical detection; F.I.D., flame ionization detection.

Magnesium ascorbyl phosphate, imidazolidinylurea, MP, EP, PP, BP, ascorbyl palmitate

Analytes

HPLC

Separation Method

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Analysis of Endocrine Disrupting Compounds in Food

and 0.02% of the total amount if the antioxidants are used in mixtures (European Communities 1995). The study of Jeong and others (2005) elucidates that high doses of BHA (500 mg/kg BW/day) induce weak dysfunction and underdevelopment of the reproductive system of male and female rats, with the change of T4 and testosterone levels, sex organ weights, sexual maturation, and histological lesions of the thyroid gland. Jobling and others (1995) revealed that chemicals present in sewage, including BHA, were weakly estrogenic. Galeano Diaz and Guiberteau Cabanillas (2004) reviewed analysis techniques of synthetic food antioxidants, including BHA in foods. These authors discuss spectrophotometric, chromatographic, and electroanalytical techniques. Continuous flow injection and UV spectrophotometric detection (DAD) have been proposed by Capitán-Vallvey and others (2004) for the simultaneous determination of the two binary mixtures, BHT/n-propyl gallate (n-PG) and BHT/BHA, in food and cosmetics samples. The method was applied to the determination of both antioxidants in fatty foods and cosmetic samples, with recoveries ranging between 101% and 105%. A flow injection method is described by Prabakar and Narayanan (2010) for the determination of the antioxidant BHA based on its catalytic oxidation at a nickel hexacyanoferrate (NiHCF) surface-modified graphite wax composite electrode that was fabricated using a new approach. The voltammetric response of BHA at the modified electrode showed current densities remarkably higher than those for the bare graphite. A detection limit of 6 × 10−7 M was obtained with a correlation coefficient of 0.9983 based on S/N = 3. Reliable results were obtained by applying the proposed flow injection method to determine BHA spiked in dehydrated potato flakes.

The influence of different parameters (working electrode, supporting electrolyte, pH, voltammetric technique) was evaluated in a quantitative simultaneous determination of three antioxidants, BHA, BHT, and TBHQ (tert-butylhydroquinone), in alcoholic mixtures and real sample foods. Glassy carbon and platinum working electrodes were investigated as mediators of oxireduction reactions. Two supporting electrolytes were investigated: Britton–Robinson 0.1 mol L−1 buffer (pH 2.0) and HCl 0.1 mol L−1 (pH 2.0), both with 2 g L−1 (p/v) of methanol. The results show that for real food samples the parameters investigated were satisfactory for quantitative determination using square wave voltammetry (SWV) without chemometric approaches and without suffering overlapping problems (dos Santos Raymundo and others 2007). An amperometric sensor based on a chemically modified electrode for the determination of BHA in food materials is described by Jayasri and Narayanan (2007). The sensor was constructed by graphite-wax composite method with manganese(II) hexacyanoferrate (MnHCF) as the electrocatalyst. Cyclic voltammetry and differential pulse voltammetry were employed to characterize the electrochemical behavior of the modified electrode. Influence of parameters such as scan rate, pH, and applied potential on the electrocatalytic response was also studied. The MnHCF-modified electrode has been successfully applied for the assay of BHA in spiked commercial samples of dry potato chips. The method can be employed for routine analysis of BHA in food samples. Phenolic antioxidants BHA, BHT, and TBHQ in lipid-containing foods were separated by GC-FID with a fused silica capillary column. (Yang and others 2002). Among 27 samples, three cooking oil, two fish oil, one butter, and one margarine were found to contain more than 200 ppm total antioxidants. The column was ChromPack CP-SIL 8CB megapore capillary column (30 m × 0.53 mm I.D., 1.5 μm film thickness).

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

A gas chromatography–mass spectrometry (GC–MS) method was developed and validated by Soliman and others (2004), allowing quantification at the nanogram per liter level of 19 analytes in water, including human pharmaceuticals, hormones, antioxidants (BHA), and a plasticizer. Extraction was an on-line continuous liquid–liquid extraction with dichloromethane. No sample cleanup or derivatization was required. The GC column was an HP-1 capillary column (12 m × 0.20 mm I.D., 0.33 μm film thickness). The application of high-performance liquid chromatography time-of-flight mass spectrometry (HPLC/TOF-MS) for the qualitative and quantitative analysis of 11 synthetic antioxidants (e.g., BHA) and preservatives in edible vegetable oil samples is reported by Li and others (2008). The recoveries at the tested concentrations of 0.1–2.0 mg/kg were 65.8–106.9. This method was suitable for routine qualitative and quantitative analyses of synthetic antioxidants and preservatives in edible vegetable oils. The column used was a reversed-phase C18 analytical column of 150 mm × 2.1 mm and 3.5 μm particle size (Zorbax Eclipse XDB-C18). Column temperature was maintained at 35°C. The injected sample volume was 20 μL. Mobile phase B was acetonitrile, and mobile phase A was water. To determine the residue levels of BHA, propyl paraben (PP), and BHT during storage, 800 kg of bulk peanuts were treated with different antioxidant emulsions. Residues were determined in peanut pod and seed tissues at 1-mo intervals during storage. The reduction levels of BHA and PP in pods at the end of the storage period ranged from 66% to 76%, and BHT levels decreased 86% (Passone and others 2008). Antioxidant content was determined by an HPLC system and UV detector (280 nm). Chromatographic separations were performed on a reversed-phase Microsorb MV 100-5, C18 column (250 mm × 4.6 mm I.D., 5 μm) and on a guard column BDSHypersil, C18 (10 mm × 4 mm ID, 5 μm). The

461

mobile phase consisted of A (methanol), B (acetonitrile), and C (acetic acid/water, 2 : 98 v/v). The detection and quantification limits, respectively, of the analytical method for the three antioxidants were 0.05 and 0.1 ng g−1 for PP, 0.76 and 1.52 ng g−1 for BHA, and 0.88 and 1.76 ng g−1 for BHT. Saad and others (2007) described an HPLC (LiChrospher reversed-phase column [250 mm × 4.0 mm length, 5 μm particle size]), with a gradient elution method for the determination of the synthetic phenolic antioxidants (SPAs) propyl gallate (PG), tertiary butyl hydroquinone (TBHQ), BHA, and BHT in foods. A C18 column served as the stationary phase; the gradient elution was formed by acetonitrile and water-acetic acid (1%). The UV detector was set at 280 nm. This method was applied to the determination of SPAs in 38 food items (16 cooking oil, 10 margarine, 6 butter, and 6 cheese samples). A gradient mode comprising ACN and water with 1% acetic acid varied between 30% and 95% over 5 min and continued to 100% in another 4 min. Next, the system was allowed to stabilize for 1–2 min under the initial conditions. The prepared mobile phase was filtered and degassed using ultrasonic agitation. Propyl gallate (PG), octyl gallate (OG), BHA, BHT, and tert-butylhydroquinone (TBHQ) are permitted in a limited number of food products according to local legislation, with individual maximum limits in each case. Perrin and Meyer (2002) describe a reversedphase HPLC method (LC-18 column ) for the quantitative determination of PG and BHA in gravies and dehydrated soups; BHA in bouillons, dehydrated meat, and dry pet food; and OG in dehydrated food. The same authors (Perrin and Meyer 2006) describe an HPLC method for the simultaneous determination of ascorbyl palmitate and synthetic phenolic antioxidants (BHA) in vegetable oils and edible fat. Lee and others (2006) evaluated SFE combined with LC–MS to determine trace preservatives and antioxidants, including

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Analysis of Endocrine Disrupting Compounds in Food

methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), BHA, BHT, α-tocopherol (α-t), and α-tocopherol acetate (α-ta) in cosmetic products. Supercritical fluid extraction was used to isolate four paraben preservatives and four antioxidants from the cosmetic matrix before quantitative analysis. The analytes were separated on a C18 reversed-phase column using methanol– water as mobile phase and were quantified by measuring their mass spectrometry. Detection limits were achieved at the level of 4.7– 142 ng/g. The synthetic antioxidants most used in oil-based food to avoid oxidation processes, BHA, BHT, and dodecyl gallate (DG), were analyzed in edible oils using micellar electrokinetic capillary chromatography (MECK) with bis-(2-ethylhexyl) sodium sulfosuccinate as the pseudostationary phase (Delgado-Zamarreño and others 2007). Studies involving solid-phase and liquid– liquid extraction were performed to find the best sample treatment before injection into the electrophoretic system. The best methodology for the isolation of antioxidants was extraction with acetonitrile from edible oil diluted with hexane. With this method BHA, BHT, and DG were evaluated at levels permitted in the European Union (European Communities 1995).

Synthetic phenolic antioxidants (SPAs) such as BHA were detected by micellar electrokinetic capillary chromatography (MECC) with electrochemical detection. This method has proved to be effective and has been successfully applied for the determination of SPA in food products (Guan and others 2006).

Styrene: Styrene dimers and trimers Styrene compounds may migrate from food containers into food. In Table 20.3, different styrene dimers and trimers are listed. The endocrine-disrupting effects of styrene monomer (SM); styrene dimers (NSD-01, -08, and -09); and styrene trimers (NST-01, -03, and -12), migrating from polystyrene (PS) containers into instant food, were investigated using in vitro and in vivo assays (Date and others 2002). Authors concluded that SM, SD, and ST exhibit no apparent estrogenic, androgenic, antiandrogenic, or thyroid activity. Styrene compounds migrated into food from disposable styrene lunch boxes (Sakamoto and others 2000) and styrene instant food containers (Kawamura and others 1998). The styrene oligomers tested were SD-1, SD-2, SD-3, SD-4, ST-1, ST-2, ST-3, ST-4, ST-5, ST-6, and ST-7.

Table 20.3. Styrene dimers and trimers. Compound Styrene dimer

Styrene trimer

Abbreviationa SD-1 SD-2 SD-3 SD-4 ST-1 ST-2 ST-3 ST-4 ST-5 ST-6 ST-7

Abbreviationb NSD-01 NSD-08 NSD-09 NST-01

NST-02 NST-03 NST-12

a b

Chemical Name 1,3-diphenylpropane 2,4-diphenyl-1-butene Cis-1,2-diphenylcyclobutane Trans-1,2-diphenylcyclobutane 2,4,6-triphenyl-1-hexene 1a-phenyl-4a-(1′phenylethyl)tetralin 1a-phenyl-4e-(1′phenylethyl)tetralin 1e-phenyl-4a-(1′phenylethyl)tetralin 1e-phenyl-4e-(1′phenylethyl)tetralin 1,3,5-triphenylcyclohexane 1e,3e,5a-triphenylcyclohexane 1-phenyl-4-(2-phenylethyl)tetralin

SD-X or ST-X numbering after Ohyama (2002) (2001). NSD-XX or NST-XX after Nobuhara and others (1999) and Azuma and others (2000).

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

Styrene dimers SD-3 and SD-4 and styrene trimers ST-1, ST-3, ST-4, and ST-5 demonstrated estrogenic activity on MCF-7 cells and binding affinity for hERα (human estrogen receptor α ) (Ohyama and others 2001). Styrene trimers ST-1, ST-3, ST-4, and ST-5 had the highest proliferative activities of the compounds tested. The relative potency of these chemicals compared to estradiol was 0.0002%–0.0015%, which was comparable with that of bisphenol A. Kadi and others (2007) studied the behavior of organic migrants originating from plastic food packages (polystyrene or polyethylene) used widely in the Saudi market. Instead of using the food itself to conduct experiments, a food simulant was used to avoid any spectral interferences. Deionized water was used as a food simulant and was made to come into contact with the polymer material under different temperatures. GC-MS analysis (capillary column, 30 m × 0.25 mm I.D., 0.25 μm thickness) was used to identify migrants. Many alkyl phthalates and hydrocarbons were found to have migrated into the food simulant. Twenty compounds were determined by GC-MS in polystyrene used for milk packaging (Abrantes 2000). The major components identified in milk were styrene and its dimer at concentrations of 13 and 43 mg/kg, respectively. A more sensitive method used to determine the concentration of styrene and its dimer in milk involved the use of acetone to precipitate the proteins and extract both the fat and the residues from the plastic packaging. The solution obtained was then directly injected into the GC. The detection limits were found to be 0.16 mg/kg for styrene and 0.28 mg/kg for the dimer. Levels of styrene monomer in foods packaged in polystyrene containers were determined by a headspace gas chromatography method and two reversed-phase highperformance liquid chromatography methods (Flanjak and Sharrad 2006). A total of 146 samples were analyzed from Victoria and

463

New South Wales, which included yogurt, cream, cheese, dessert, ice cream, egg white, onion dip, and margarine. The highest level of styrene found was 0.1 mg kg−1 in yogurt. About 85% of all yogurt samples were found to have values less than 0.05 mg kg−1. The lowest values of styrene obtained were for margarine samples, of which more than 90% contained less than 0.010 mg kg−1. The objective of the study (Guillot and others 2006) was to compare the official EU liquid–liquid extraction method (EPA 2003) with SPME for the analysis of compounds, such as styrene, migrating from cross-linked polyethylene into drinking water. A PDMS/ DVB 65-μm fiber was most efficient. Optimum extraction conditions were an immersion time of 30 min and heating to 60°C. In the paper of Carillo-Carrión and others (2007), the use of surfactant-coated multiwalled carbon nanotubes as additive in liquid–liquid extraction was applied for the determination of BTEX-S compounds (S = styrene) in olive oil samples. After sample treatment, the aqueous extracts were subsequently analyzed by headspace/gas chromatography/mass spectrometry allowing the determination of BTEX-S within approximately 15 min. The presence of styrene was directly related to the packaging material, the concentration of styrene being higher when the samples were stored in plastic bottles: a concentration between 30 and 100 ng/mL. A relationship between styrene concentration and the storage time was also observed. Samples bottled in 2006 contained a lower concentration of styrene that those with a longer packing time. Marsin Sanagi and others (2008) describe a method for quantitative determination of VOCs, namely styrene, toluene, ethylbenzene, iso-propylbenzene, and n-propylbenzene that migrated from polystyrene food packaging into food simulant, by GC-FID. Headspace solid-phase microextraction (HSSPME) was applied for a migration test using water as food simulant. The effects of

464

Analysis of Endocrine Disrupting Compounds in Food

extraction variables, including sample volume, eluotropic strength, extraction temperature, extraction time, desorption time, sample agitation, and salt addition on the amounts of the extracted analytes were studied to obtain the optimal HS-SPME conditions. The optimized method was applied to test the VOCs that migrated from polystyrene bowls and cups at storage temperatures ranging from 24°C to 80°C for 30 min. Styrene and ethylbenzene were found to migrate from the samples into the food simulant. The migration of analyte was found to be strongly dependent upon the storage temperature. Ten samples of yogurt in PS containers were analyzed for styrene monomer content (Withey 1976). These yogurt containers were purchased locally, and no record of their history, apart from the recommended date of expiration, was available. A 1-g portion of the yogurt was placed in a 15-mL septum vial and shaken for 30 min at 60°C. The headspace phase was injected onto the gas chromatograph (column, 6 ft × 0.125 I.D., Carbowax 400 on Porasil F). The limit of detection was about 1 ppb (w/v) and styrene monomer contents varied between 2.5 and 34.6 ppb (mean 12 ppb). The same method was used for the evaluation of styrene monomers in different Canadian foods such as yogurt, milk, butter fat cream, cottage cheese, sour cream, jellified milk, and honey (Withey and Collins 1978). Styrene monomer contents varied between 0 and 245.2 ppb. Concentrations increased with time. The release of ethylbenzene and styrene from plastic cheese containers was monitored by SPME-GC-MS (Chiesa and others 2008). Watanabe-Suzuki and others (2001) determined styrene and related products in human body fluid by HS-CGC (headspace–capillary gas chromatography) with cryogenic oven trapping. The column used was Rtx-Volatile fused silica middle-bore capillary column (30 × 0.32 mm I.D., 1.5 μm).

BTEX-S residues can be present in virgin oils either naturally or as contaminants (Peña and others 2004). By combining HS-GC with MS, Peña was able to confirm the presence of BTEX-S residues in oils. The column was a HP-5MS fused silica capillary column (45 m × 0.32 mm I.D., 0.25 μm film thickness). Concentrations of styrene ranged from not detectable to 620 ng/mL. Volatiles of olive oils from tree-picked, ground-picked, and tree-picked and groundpicked combined olives were analyzed by solid-phase microextraction and gas chromatography (HP5 column, 30 m × 0.25 mm I.D., 0.25 μm film thickness) with FID (flame ionization detection) (Jiménez and others 2006). Headspace sampling was carried out for 30 min with a 74-μm PDMS/DVB fiber exposed at 40°C of sampling temperature. Chromatographic data were analyzed by principal components analysis (PCA). Compounds such as 4-ethylphenol and styrene were identified by gas chromatography–mass spectrometry (GC-MS) in ground-picked olive oils. Usually, styrene is associated with oil from fruits stored in plastics. However, it can occur naturally in some foods, because cinnamic acids present in the phenol fraction of virgin olive oil can be converted to styrene by enzymes.

References Abrantes S. GC-MS identification of styrene and oligomers in polystyrene plastic for milk packaging. Headspace and solution injection technique. J. High Resolut. Chromatogr. 16(2):113–115, 2000. Adolfsson-Erici M., Pettersson M., Parkkonen J., Sturve J. Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden. Chemosphere. 46(9–10):1485–1489, 2002. Akhtar M.J., Khan S., Roy I.M., Jafri I.A. High performance liquid chromatographic determination of phenoxetol, methyl paraben, ethyl paraben, n-propyl paraben, iso-butyl paraben, n-butyl paraben and croconazole HCl. J. Pharm. Biomed. Anal. 14(11):1609– 1613, 1996. Allmyr M., Adolfsson-Erici M., McLachlan M.S., Sandborgh-Englund G. Determination of triclosan as its pentafluorobenzoyl ester in human plasma and milk using electron capture negative ionization mass

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

spectrometry. Sci. Total Environ. 372(1):87–93, 2006a. Allmyr M., McLachlan M.S., Sandborgh-Englund G., Adolfsson-Erici M. Determination of triclosan as its pentafluorobenzoyl ester in human plasma and milk using electron capture negative ionization mass spectrometry. Anal. Chem. 78(18):6542–6546, 2006b. Basheer C., Lee H.K. Analysis of endocrine disrupting alkylphenols, chlorophenols and bisphenol-A using hollow fiber-protected liquid-phase microextraction coupled with injection port-derivatization gas chromatography–mass spectrometry. J. Chromatogr. A. 1057(1–2):163–169, 2004. Blanco E., del Carmen Casais M., del Carmen Mejuto M., Cela R. Combination of off-line solid-phase extraction and on-column sample stacking for sensitive determination of parabens and p-hydroxybenzoic acid in waters by non-aqueous capillary electrophoresis. Anal. Chim. Acta. 647(1):104–111, 2009. Burini G. Determination of the alkyl esters of phydroxybenzoic acid in mayonnaise by highperformance liquid chromatography and fluorescence labelling. J. Chromatogr. A. 664(2):213–219, 1994. Butte W., Fooken C. Simultaneous determination of pentachlorphenol and neutral organochlorine compounds in human milk. Fresenius J. Anal. Chem. 336:511– 514, 1990. Campillo N., Peñalver R., Hernández-Córdoba M. Evaluation of solid-phase microextraction conditions for the determination of chlorophenols in honey samples using gas chromatography. J. Chromatogr A. 1125(1):31–37, 2006. Canosa P., Pérez-Palacios D., Garrido-López A., Tena M.T., Rodríguez I., Rubí E., Cela R. Pressurized liquid extraction with in-cell clean-up followed by gas chromatography-tandem mass spectrometry for the selective determination of parabens and triclosan in indoor dust. J. Chromatogr A. 1161(1–2):105–112, 2007. Capitán-Vallvey L.F., Valencia M.C., Arana Nicolás E. Solid-phase ultraviolet absorbance spectrophotometric multisensor for the simultaneous determination of mutilated hydroxytoluene and co-existing antioxidants. Anal. Chim. Acta. 503(2):179–186, 2004. Carabias-Martínez R., Rodríguez-Gonzalo E., RevillaRuiz P. Determination of endocrine-disrupting compounds in cereals by pressurized liquid extraction and liquid chromatography-mass spectrometry: Study of background contamination. J. Chromatogr A. 1137(2): 207–215, 2006. Carabias-Martínez R., Rodríguez-Gonzalo E., RevillaRuiz P. Determination of weakly acidic endocrinedisrupting compounds by liquid chromatographymass spectrometry with post-column base addition. J. Chromatogr A. 1056(1–2):131–138, 2004. Carrillo-Carrión C., Lucena R., Cárdenas S., Valcárcel M. Liquid-liquid extraction/headspace/gas chromatographic/mass spectrometric determination of benzene, toluene, ethylbenzene, (o-, m- and p-)xylene and styrene in olive oil using surfactant-coated carbon nanotubes as extractant. J. Chromatogr A. 1171(1– 2):1–7, 2007.

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Castillo M., Alpendurada M.F., Barceló D. Characterization of organic pollutants in industrial effluents using liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. J. Mass Spectrom. 32(10):1100–1110, 1997. Chiesa L.M., Soncin S., Panseri S., Cantoni C. Release of ethylbenzene and styrene from plastic cheese containers. Vet. Res. Commun. 32(suppl 1):319–321, 2008. Chu T.Y., Chen C.L., Wang H.F. A rapid method for the simultaneous determination of preservatives in soy sauce. J. Food Drug Anal. 11(3):246–250, 2003. Cooper J.F., Tourte J., Gros P. Determination of pentachlorophenol residues in wine and corks by solvent extraction methodology and specific gas chromatography detection. Chromatographia. 38(3–4):147–150, 1994. Darbre P.D., Aljarrah A., Miller W.R., Coldham N.G., Sauer M.J., Pope G.S. Concentrations of parabens in human breast tumours. J. Appl. Toxicol. 24(1):5–13, 2004. Darbre P.D., Harvey P.W. Paraben esters: Review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks. J. App. Toxicol. 28(5):561–578, 2008. Date K., Ohno K., Azuma Y., Hirano S., Kobayashi K., Sakurai T., Nobuhara Y., Yamada T. Endocrinedisrupting effects of styrene oligomers that migrated from polystyrene containers into food. Food Chem Toxicol. 40(1):65–75, 2002. David F., Sandra P. Stir bar sorptive extraction for trace analysis. J. Chromatogr. A. 1152(1–2):54–69, 2007. Dayan A.D. Risk assessment of triclosan [Irgasan®] in human breast milk. Food and Chemical Toxicology. 45(1):125–129, 2007. De Croo F., De Schutter J., Van den Bossche W., De Moerloose P. Gas chromatographic determination of parabens in various pharmaceutical dosage forms. Chromatographia. 18(5):260–264, 1984. Delgado-Zamarreño M.M., González-Maza I., SánchezPérez A., Carabias Martínez R. Analysis of synthetic phenolic antioxidants in edible oils by micellar electrokinetic capillary chromatography. Food Chemistry. 100(4):1722–1727, 2007. Dorsey W.C., Tchounwou P.B. Pentochlorophenolinduced cytotoxic, mitogenic, and endocrinedisrupting activities in channel catfish, Ictalurus punctatus. Int. J. Env. Res. Publ. Health. 1(2):90–99, 2004. dos Santos Raymundo M., Marques da Silva Paula M., Franco C., Fett R. Quantitative determination of the phenolic antioxidants using voltammetric techniques. LWT - Food Sci. Tech. 40(7):1133–1139, 2007. Driouich R., Takayanagi T., Oshima M., Motomizu S. Separation and determination of haloperidol, parabens and some of their degradation products by micellar electrokinetic chromatography. J. Chromatogr. A. 903(1–2):271–278, 2000. EFSA (European Food Safety Authority). EFSA-Q2009-411. 2009. Available at: http://www.efsa.europa.eu/ en/scdocs/scdoc/1104.htm. Eurofins. Consumer product testing in Hamburg offers analyses to securely detect 4-methylbenzophenone,

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Analysis of Endocrine Disrupting Compounds in Food

benzophenone and other UV-initiators in packaging materials and food. Available at: http://producttesting.eurofins.com/news/announcements–updates/ new-analyses-for-detection-of-4-methylbenzophenone, - benzophenone - and - other- uv - initiators.aspx Accessed in February, 2009. European Commission, SANCO D3/LR. Provisional list of monomers and additives notified to European Commission as substances which may be used in the manufacture of plastics intended to come into contact with foodstuffs (updated to 25 July 2003), 2003. Available at: http://ec.europa.eu/food/fs/sfp/food_ contact/note_guidance_en.pdf. European Commission, SCCP/0873/05, Scientific Committee on Consumer Products—SCCP. Extended Opinion on the Safety Evaluation of Parabens, 2005. Available at: ec.europa.eu/health/ph_risk/committees/ …/sccp_o_019.pdf. European Communities, European Parliament and Council. Directive 95/2/EC of 20 February 1995 on food additives other than colours and sweeteners. Off. J. Eur. Comm., 1995. Fattahi N., Samadi S., Assadi Y., Hosseini M.R.M. Solidphase extraction combined with dispersive liquidliquid microextraction-ultra preconcentration of chlorophenols in aqueous samples. J. Chromatogr. A. 1169(1–2):63–69, 2007. Felix T., Hall B.J., Brodbelt J.S. Determination of benzophenone-3 and metabolites in water and human urine by solid-phase microextraction and quadrupole ion trap GC-MS. Anal. Chim. Acta. 371(2–3):195– 203, 1998. Flanjak J., Sharrad J. Quantitative analysis of styrene monomer in foods. A limited East Australian Survey. J. Sci. Food Agric. 35(4):457–462, 2006. Galeano Diaz T., Guiberteau Cabanillas A. Analysis of synthetic food antioxidants. In: Handbook of Food Analysis, 2nd ed., L.M.L. Nollet, ed. Marcel Dekker, New York, 2004. García-Jiménez J.F., Valencia M.C., Capitán-Vallvey L.F. Simultaneous determination of antioxidants, preservatives and sweetener additives in food and cosmetics by flow injection analysis coupled to a monolithic column. Anal. Chim. Acta. 594(2):226– 233, 2007. Giokas D.L., Sakkas, V.A., Albanis T.A. Determination of residues of UV filters in natural waters by solidphase extraction coupled to liquid chromatographyphotodiode array detection and gas chromatography-mass spectrometry. Anal. Chim. Acta. 477(2):257–267, 2004. Giokas D.L., Salvador A., Chisvert A. UV filters: From sunscreens to human body and the environment. Trends Anal. Chem. 26(5):360–374, 2007. Golden R., Gandy J., Vollmer G. A review of the endocrine activity of parabens and implications for potential risks to human health. Crit. Rev. Toxicol. 35(5):435–458, 2005. Guan Y., Chu Q., Fu L., Wu T., Ye J. Determination of phenolic antioxidants by micellar electrokinetic capillary chromatography with electrochemical detection. Food Chem. 94(1):157–162, 2006.

Guillarme D., Rudaz S., Schelling C., Dreux M., Veuthey J.L. Micro liquid chromatography coupled with evaporative light scattering detector at ambient and high temperature: Optimization of the nebulization cell geometry. J. Chromatogr. A. 1192(1):103–112, 2008. Guillot S., Kelly M.T., Fenet H., Larroque M. Evaluation of solid-phase microextraction as an alternative to the official method for the analysis of organic micropollutants in drinking water. J. Chromatogr. A. 101(1– 2):46–52, 2006. Hajkova R., Solich P., Pospisilova M., Sicha J. Simultaneous determination of methylparaben, propylparaben, sodium diclofenac and its degradation product in a topical emulgel by reversed-phase liquid chromatography. Anal. Chim. Acta. 467(1–2):91–96, 2002. Heo Y.H., Lee K.J. Application of micellar electrokinetic capillary chromatography for the determination of benzoic acid and its esters in liquid formula medicines as preservatives. J. Pharmac. Biomed. Anal. 17(8):1371–1379, 1998. Insa S., Salvadó V., Anticó E. Development of solidphase extraction and solid-phase microextraction methods for the determination of chlorophenols in cork macerate and wine samples. J. Chromatogr. A. 1047(1):15–20, 2004. Insa S., Salvadó V., Anticó E. Assays on the simultaneous determination and elimination of chloroanisoles and chlorophenols from contaminated cork samples. J. Chromatogr. A. 122(1–2):215–221, 2006. Jayasri D., Sriman Narayanan S. Manganese(II) hexacyanoferrate based renewable amperometric sensor for the determination of butylated hydroxyanisole in food products. Food Chem. 101(2):607–614, 2007. Jeon H.K., Chung Y., Ryu J.C. Simultaneous determination of benzophenone-type UV filters in water and soil by gas chromatography-mass spectrometry. J. Chromatogr. A. 1131(1–2):192–202, 2006. Jeong S.H., Kim B.Y., Kang H.G., Ku H-O., Cho J.H. Effects of butylated hydroxyanisole on the development and functions of reproductive system in rats. Toxicology. 208(1):49–52, 2005. Jiménez A., Aguilera M.P., Beltrán G., Uceda M. Application of solid-phase microextraction to virgin olive oil quality control. J. Chromatogr. A. 1121(1):140–144, 2006. Jobling S., Reynolds T., White R., Parker M.G., Sumpter J.P. A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ. Health Perspect. 103(6) 582–587,1995. Kadi M.W., Ismail I.M., Sobahi T.R. Identification of organic migrants from some plastic food packaging used in Saudi Arabia using water as food simulant. Chem. Asian J. 19(4):2744–2750, 2007. Kang S.H., Kim H. Simultaneous determination of methylparaben, propylparaben and thimerosal by high-performance liquid chromatography and electrochemical detection. J. Pharm. Biomed. Anal. 15(9– 10):1359–1364, 1997. Kawaguchi M., Inoue K., Yoshimura M., Sakui N., Okanouchi N., Ito R., Yoshimura Y., Nakazawa H.

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

Trace analysis of phenolic xenoestrogens in water samples by stir bar sorptive extraction with in situ derivatization and thermal desorption-gas chromatography-mass spectrometry. J. Chromatogr A. 1041(1–2):19–26, 2004. Kawamura Y., Nishi K., Maehara T., Yamada. T. Migration of styrene dimers and trimers from polystyrene containers into instant foods. J. Food Hyg. Soc. Jpn. 39:390–398, 1998. Kulikov A.U., Verushkin, A.G. Simultaneous determination of paracetamol, caffeine, guaifenesin and preservatives in syrups by micellar L.C. Chromatographia. 67:347–355, 2008. Labat L., Kummer E., Dallet P., Dubost J.P. Comparison of high-performance liquid chromatography and capillary zone electrophoresis for the determination of parabens in a cosmetic product. J. Pharm. Biomed. Anal. 23(4):763–769, 2000. Lambropoulou D.A., Konstantinou I.K., Albanis T.A. Recent developments in headspace microextraction techniques for the analysis of environmental contaminants in different matrices. J. Chromatogr A. 1152(1– 2):70–96, 2007. Lee M-R., Lee C-Y., Li Z.G., Tsai T.F. Simultaneous analysis of antioxidants and preservatives in cosmetics by supercritical fluid extraction combined with liquid chromatography-mass spectrometry. J. Chromatogr A. 1120(1–2):244–251, 2006. Li X.Q., Zhang F., Sun Y.Y., Yong W., Chu X.G., Fang Y.Y., Zweigenbaum J. Accurate screening for synthetic preservatives in beverage using high performance liquid chromatography with time-of-flight mass spectrometry. Anal. Chim. Acta. 608(2):165– 177, 2008. Llompart M., Lourido M., Landín P., García-Jares C., Cela R. Optimization of a derivatization-solid-phase microextraction method for the analysis of thirty phenolic pollutants in water samples. J. Chromatogr. A. 963(1–2):137–148, 2002. Lokhnauth J.K., Snow N.H. Determination of parabens in pharmaceutical formulations by solid-phase microextraction-ion mobility spectrometry. Anal. Chem. 77(18):5938–5946, 2005. Maeda Y., Yamamoto M., Owada K., Sato S., Masui T., Nakazawa H., Fujita M. High-performance liquid chromatographic determination of six phydroxybenzoic acid esters in cosmetics using SepPak florisil cartridges for sample pre-treatment. J. Chromatogr. A. 410(2):413–418, 1987. Mardones C., von Baer D., Silva J., Retamal M.J. Determination of halophenolic wood preservant traces in milk using headspace solid-phase microextraction and gas chromatography–mass spectrometry. J. Chromatogr A. 1215(1–2):1–7, 2008. Marsin Sanagi M., Lu S.L., Zalilah N., Wan A., Wan I., Ahmedy A.N. Determination of residual volatile organic compounds migrated from polystyrene food packaging into food simulant by headspace solid phase microextraction-gas chromatography. Malaysian J. Anal. Sci. 12(3):542–551, 2008. Martínez-Uruñuela A., González-Sáiz J.M., Pizarro C. Multiple solid-phase microextraction in a non-

467

equilibrium situation: Application in quantitative analysis of chlorophenols and chloroanisoles related to cork taint in wine. J. Chromatogr. A. 1089(1–2):31– 38, 2005. Martínez-Uruñuela A., González-Sáiz J.M., Pizarro C. Optimisation of the derivatisation reaction and subsequent headspace solid-phase microextraction method for the direct determination of chlorophenols in red wine. J. Chromatogr. A. 1048(2):141–151, 2004. Miller D., Wheals B.B., Beresford N., Sumpter J.P. Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay. Environ. Health Perspect. 109(2):133–138, 2001. Moloney F.J., Collins S., Murphy G.M. Sunscreens: Safety, efficacy and appropriate use. Am. J. Clin. Dermatol. 3(3):185–191, 2002. Myint A., Zhang Q., Liu L., Cui H. Flow injectionchemiluminescence determination of paraben preservative in food safety. Anal. Chim. Acta. 517(1–2): 119–124, 2004. Negreira N., Rodriguez I., Ramil M., Rubi E., Cela R. Sensitive determination of salicylate and benzophenone type UV filters in water samples using solidphase microextration, derivatization and gas chromatography tandem mass spectrometry. Anal. Chim. Acta. 638(1):36–44, 2009. Núñez L., Tadeo J.L., García-Valcárcel A.I., Turiel E. Determination of parabens in environmental solid samples by ultrasonic-assisted extraction and liquid chromatography with triple quadrupole mass spectrometry. J. Chromatogr. A. 1214(1–2):178–182, 2008. Ohyama, K., Nagai F., Tsuchiya Y. Certain styrene oligomers have proliferative activity on MCF-7 human breast tumor cells and binding affinity for human estrogen receptor. Environ. Health Perspect. 109:699–703, 2001. Okereke C.S., Barat S.A., Abdel-Rahman M.S. Safety evaluation of benzophenone-3 after dermal administration in rats. Toxicol. Lett. 80(1–3):61–67, 1995. Okereke C.S., Kadry A.M., Abdel-Rahman M.S., Davis R.A., Friedman M.A. Metabolism of benzophenone-3 in rats. Drug Metab. Dispos. 21(5): 788–791, 1993. Orton F., Lutz I., Kloos W., Rouletge E.J. Endocrine disrupting effects of herbicides and pentochlorophenol: in vitro and in vivo evidence. Envron. Sci. Technol. 43(6):2144–2150, 2009. Ozaki A., Yamaguchi Y., Fujita T., Kuroda K., Endo G. Chemical analysis and genotoxicological safety assessment of paper and paperboard used for food packaging. Food Chem. Toxicol. 42(8):1323–1337, 2004. Passone M.A, Funes G.J., Resnik S.L., Etcheverry M.G. Residue levels of food-grade antioxidants in postharvest treated in-pod peanuts during five months of storage. Food Chem. 106(2):691–697, 2008. Pedrouzo M., Borrull F., Marcé R.M., Pocurull E. Ultrahigh-performance liquid chromatography-tandem mass spectrometry for determining the presence of eleven personal care products in surface and waste waters. J. Chromatogr. A. 1216(42):6994–7000, 2009.

468

Analysis of Endocrine Disrupting Compounds in Food

Peña F., Cardenas S., Gallego M., Valcarel M. Combining headspace gas chromatography with mass spectrometry detection for confirmation of hydrocarbon residues in virgin oil following automatic screening. J. Chromatogr. A. 1052(1–2):137–143, 2004. Perrin C., Meyer L. Quantification of synthetic phenolic antioxidants in dry foods by reversed-phase HPLC with photodiode array detection. Food Chem. 77(1):93–100, 2002. Perrin C., Meyer L. Simultaneous determination of ascorbyl palmitate and nine phenolic antioxidants in vegetable oils and edible fats by HPLC. J. Am. Oil Chem. Soc. 80(2):115–118, 2006. Pizarro C., Martínez-Uruñuela A., Pérez-del-Notario N., González-Sáiz J.M. Robustness test of a headspace solid-phase microextraction method for the determination of chloroanisoles and chlorophenols related to cork taint in wine using experimental design. J. Chromatogr. A. 1208(1–2):54–61, 2008. Pizarro C., Pérez-del-Notario N., González-Sáiz J.M. Optimisation of a microwave-assisted extraction method for the simultaneous determination of haloanisoles and halophenols in cork stoppers. J. Chromatogr. A. 1149(2):138–144, 2007a. Pizarro C., Pérez-del-Notario N., González-Sáiz J.M. Optimisation of a headspace solid-phase microextraction with on-fiber derivatisation method for the direct determination of haloanisoles and halophenols in wine. J. Chromatogr. A. 1143(1–2):26–35, 2007b. Polo M., Llompart M., Garcia-Jares C., Cela R. Development of a solid-phase microextraction method for the analysis of phenolic flame retardants in water samples. J. Chromatogr. A. 1124(1–2):11–21, 2006. Prabakar S.J.R., Narayanan S.S. Flow injection analysis of BHA by NiHCF modified electrode. Food Chem. 118(2):449–455, 2010. Regueiro J., Becerril E., Garcia-Jares C., Llompart M. Trace analysis of parabens, triclosan and related chlorphenols in water by headspace solid-phase microextraction with in situ derivatization and gas chromatography-tandem mass spectrometry. J. Chromatogr. A. 1216(1–2):4693–4702, 2009. Rieger M.M. Photostability of cosmetic ingredients on the skin: Disposal of energy by photoactive molecules in sunscreens can adversely affect the sunscreens’ function and safety. Cosmetics & Toiletries 112(6):65– 72, 1997. Rodil R., Quintana J.B., Lopez-Mahia P., MuniateguiLorenzo S., Prada-Rodriguez D. Multi-residue analytical method for the determination of emerging pollutants in water by solid-phase extraction and liquid chromatography-tandem mass spectrometry. J. Chromatogr. A. 1216(14):2958–2969, 2009. Routledge E.J., Parker J., Odum J., Ashby J., Sumpter J.P. Some alkyl hydroxy benzoate preservatives (parabens) are estrogenic. Toxicol. Appl. Pharm. 153(1):12–19, 1998. Rudel R.A., Camann D.E., Spengler J.D., Korn L.R., Brody J.G. Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other endocrinedisrupting compounds in indoor air and dust. Environ. Sci. Technol. 37(20):4543–4553, 2003.

Saad B., Bari M.F., Saleh M.I., Ahmad K., Talib M.K.M. Simultaneous determination of preservatives (benzoic acid, sorbic acid, methylparaben and propylparaben) in foodstuffs using high-performance liquid chromatography. J. Chromatogr. A. 1073(1–2):393–397, 2005. Saad B., Yong Y.S., Mohd A.N., Noor H.H., Abdussalam Salhin M.A., Muhammad I.S., Shaida F.S., Khairuddin M.T., Kamarudzaman A. Determination of synthetic phenolic antioxidants in food items using reversedphase HPLC. Food Chem. 105(1):389–394, 2007. Sagratini G., Capriolo G., Cristalli G., Giardina D., Ricciutelli M., Volpini R., Zuo Y., Vittori S. Determination of ink photoinitiators in packaged beverages by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. J. Chromatogr. A. 1194(2):213–220, 2008. Sakamoto H., Matsuzawa A., Itoh R., Tohyama Y. Quantitative analysis of styrene dimer and trimers migrated from disposable lunch boxes. J. Food Hyg. Soc. Jpn. 41:200–205, 2000. Salafranca J., Domeño C., Fernández C., Nerín C. Experimental design applied to the determination of several contaminants in Duero River by solid-phase microextraction. J. Chromatogr. A. 1026(1–2):289– 293, 2003. Schlumpf M., Cotton B., Conscience M., Haller V., Steinmann B., Lichtensteiger W. In vitro and in vivo estrogenicity of UV screens. Environ. Health Perspect. 109(3):239–244, 2001. Silva E., Rajapakse N., Kortenkamp A. Something from “nothing”—eight week estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. Environ. Sci. Technol. 36(8): 1751–1756, 2002. Soliman M.A., Pedersen J.A., Suffet I.H. Rapid gas chromatography–mass spectrometry screening method for human pharmaceuticals, hormones, antioxidants and plasticizers in water. J. Chromatogr. A. 1029(1–2):223–237, 2004. Soni M.G., Carabin I.G., Burdock G.A. Safety assessment of esters of p-hydroxybenzoic acid (parabens). Food Chem. Toxicol. 43(57):985–1015, 2005. Sottofattori E., Anzaldi M., Balbi A., Tonello G. Simultaneous HPLC determination of multiple components in a commercial cosmetic cream. J. Pharm. Biomed. Anal. 18:213–217, 1998. Takatori S., Kitagawa Y., Oda H., Miwa G., Nishikawa J., Nishihara T., Nakazawa H., Hori S. Estrogenicity of metabolites of benzophenone derivatives examined by a yeast two-hybrid assay. J. Health Sci. 49(2):91– 98, 2003. U.S. EPA. Clean Water Act Analytical Methods. Available at: www.epa.gov/ost/methods/. 2003. Accessed June 20, 2010. The Council Directive 76/768/EC, 1976. Volume 1. Cosmetics Legislation, Cosmetic Products. Available at: http://www.leffingwell.com/cosmetics/vol_1en.pdf. Accessed June 20, 2010. Veningerova M., Uhnak J., Prachar V., Kovacicova J. Chlorinated phenols in human milk. Z. Lebensm. Unters. Forsch. 203:309–310, 1996.

Pentachlorophenol, Benzophenone, Parabens, Butylated Hydroxyanisole, and Styrene

Wang L., Zhang X., Wang Y., Wang W. Simultaneous determination of preservatives in soft drinks, yogurts and sauces by a novel solid-phase extraction element and thermal desorption-gas chromatography. Anal. Chim. Acta. 577(1):62–67, 2006. Wang S.P., Chang C.L. Determination of parabens in cosmetic products by supercritical fluid extraction and capillary zone electrophoresis. Anal. Chim. Acta. 377 (1):85–93, 1998. Watanabe-Suzuki K., Ishii A., Seno H., Takeuchi Y., Suzuki O. Sensitive determination of styrene and related compounds in human body fluids by headspace capillary gas chromatography with cryogenic oven trapping. Chromatographia. 54(7–8): 507–510, 2001. Withey J.R., Collins P.G. Styrene monomer in foods: A limited Canadian survey. Bull. Environ. Contam. Toxicol. 19(1):86–94, 1978. Withey J.R. Quantitative analysis of styrene monomer in polystyrene and foods including some preliminary studies of the uptake and pharmacodynamics of the monomer in rats. Environ. Health Perspect. 17:125– 133, 1976. Xiu-Qin L., Chao J., Wei Y., Yun L., Min-Li Y., XiaoGang C. UPLC-DAD analysis for simultaneous determination of ten synthetic preservatives in foodstuff. Chromatographia. 68(1–2):57–63, 2008.

469

Xiu-Qin L., Chao J., Yan-Yan S., Min-Li Y., Xiao-Gang C. Analysis of synthetic antioxidants and preservatives in edible vegetable oil by HPLC/TOF-MS. Food Chem. 113(2):692–700, 2009. Yang M.H., Lin H.J., Choong Y.M. A rapid gas chromatographic method for direct determination of BHA, BHT and TBHQ in edible oils and fats. Food Res. Int. 35(7):627–633, 2002. Ye X., Bishop A.M., Needham L.L., Calafat A.M. Automated on-line column-switching HPLC-MS/MS method with peak focusing for measuring parabens, triclosan, and other environmental phenols in human milk. Anal. Chim. Acta. 622(1–2):150–156, 2008. Zalacain A., Alonso G.L., Lorenzo C., Iñiguez M., Salinas M.R. Stir bar sorptive extraction for the analysis of wine cork taint. J. Chromatogr. 1033(1):173– 178, 2004. Zhang Q., Lian M., Liu L., Cui H. High-performance liquid chromatographic assay of parabens in wash-off cosmetic products and foods using chemiluminescence detection. Anal. Chim. Acta. 537(1–2):31–39, 2005. Zhou F., Li X., Zeng Z. Determination of phenolic compounds in wastewater samples using a novel fiber by solid-phase microextraction coupled to gas chromatography. Anal. Chim. Acta. 538(1–2):63–70, 2005.

Index

AAS. See Atomic absorption spectrometry Absorption columns, 31–32 Acaricides, classification, 129 Accelerated solvent extraction (ASE), 26–27 alkylphenolic compounds in foodstuffs sample extraction with, 309–14 ED pesticides from solid food matrices and, 89 hormones in food, 247 OT compound analysis, 271 PBB analysis and, 336 sample drying for, 26–27 sample purification for, 27 solvents for, 26 Acephate, 199 Acetone, 110, 246, 261 Acetonitrile, 110, 246 Acetylation, GC and, 250–51 Acetylcholinesterase (AChE) inhibition test, 212–13 AChE inhibition test. See Acetylcholinesterase inhibition test Acid digestion, 32 Adrenal glands, 5 Adsorptive stripping voltammetry (AdSV), 182 AdSV. See Adsorptive stripping voltammetry AES. See Atomic emission spectrometry AFS. See Atomic fluorescence spectrometry AhR. See Aryl hydrocarbon receptor Aldrin, 79 Alkphenolic compounds, endocrine-disrupting activities of, 306–7 Alkylphenol ethoxylates (APEOs), 305–6 degradation pathway, 306, 306f in food, 307 in seafood, 307 Alkylphenolic compounds in food matrices, 307–8

Analysis of Endocrine Disrupting Compounds in Food Edited by Leo M. L. Nollet © 2011 Blackwell Publishing, Ltd. ISBN: 978-0-813-81816-0

in foodstuffs, analysis of, 308–20, 309f, 310t analytes and, 308 ASE for, 309–14 background contamination and, 317 column chromatography for, 316 detection of, 317–18 GC-MS for, 318 GPC for, 316 HPLC for, 317 LC for, 319 LC-MS for, 319–20 matrices and, 308 MSPDE for, 315 purification for, 316–17 sample extraction and, 308–16 solvents and sample extraction for, 308–9 SOX for, 314 SPE for, 315, 316–17 SPME for, 315–16 steam distillation for, 314–15 UAE for, 309 Alkyl phenols, 7 Alkylphenols (APs), 305–6 aquatic pollution with, 308 in food, 307 in seafood, 307 Alumina, 24 Ambient ionization MS, 214 Ambient MS, OP pesticide analysis with, 211, 213–14 AMDIS. See Automated mass spectral deconvolution and identification system Amino acid derivatives, 5 Anhydrous sodium sulfate, 248 Animal tissues EDCs in, 130 PCBs in, 25 Antiandrogens, environmental, 7 Antiestrogens, environmental, 7. See also Estrogen(s) Antihelmintics, 199 471

472

Index

Antioxidants. See also Preservatives/antioxidants phenolic, 460 synthetic, 461–62 Antiseptics. See Disinfectants/antiseptics APCI. See Atmospheric pressure chemical ionization APEOs. See Alkylphenol ethoxylates APGD-MS. See Atmospheric pressure glow discharge mass spectrometry API. See Atmospheric pressure ionization APPI. See Atmospheric pressure photoionization APs. See Alkylphenols Aquatic environments. See also Environment APs and pollution of, 308 DL-PCBs in, 40 surfactants and toxicity in, 305 Arsenic, in food, 302 Aryl hydrocarbon receptor (AhR), 19 activation, 41–42 DL-PCB binding to, 41 AS. See Atomic spectroscopy ASE. See Accelerated solvent extraction Atmospheric pressure chemical ionization (APCI), 95 Atmospheric pressure glow discharge mass spectrometry (APGD-MS), 214 Atmospheric pressure ionization (API), 95 Atmospheric pressure photoionization (APPI), 95 Atomic absorption spectrometry (AAS), 290 atomizer, 291–93 filament, 292–93 rod, 292 ETA, 292 heavy metals in food determined with, 290–93 instrumentation, 291 theory, 290–91 Atomic emission spectrometry (AES), 290 CCD for, 294–95 CID for, 294–95 CTD for, 294–95 heavy metals in food determined by, 293–96 high-resolution monochromator, 294 ICP for, 295–96, 295f instrumentation, 294–96 PDAs for, 294 plasma sources, 295–96 theory, 293–94 Atomic fluorescence spectrometry (AFS), 296–97

Atomic spectroscopy (AS) analytical practice of, 298–99 techniques, 298 heavy metals in food, determined by, 289–303 sample preparation for, 299 selected applications of, 299–303, 300t methods, 289–90 Automated mass spectral deconvolution and identification system (AMDIS), 113 Bactericides, classification, 129 BaP. See Benzo[α]pyrene BDMs. See Bioanalytical detection methods Benzo[α]pyrene (BaP) in food, 430–31, 430t in food, occurrence of, 441–42, 441t preseparation procedures, 433 GC, 434 Benzophenone (BP), 447, 451–53, 451t analysis methods, 452–53 risk assessment, 452 sample preparation methods, 452 uses, 451 BFRs. See Brominated flame retardants BHA. See Butylated hydroxyanisole Bioanalytical detection methods (BDMs), 68 Bioanalytical screening methods, 68–69 Bioassays, 39–40 Bio-Beads SX3, 31 Biochanin A, 223 Biomagnification, 402–3 Biosensors AChE inhibition and, 213–14 disposable, 212 electrochemical, 212 enzyme, 212 OP pesticide analysis with, 211, 212–13 Birds EDCs effects on, 11 PBBs and, 340–41 Bisphenol A (BPA), 10, 349–63 analytical methods, 349–50, 350f chemical structure, 349, 350f detection, 357–62, 358t ED, 360 fluorescence, 360 GC-MS, 361–62 immunochemical methods, 362 LC, 360 MS, 361

Index

estrogenic activity, 349 in food, 351t market, 349 removal of background contamination and, 350–52 sample cleanup, 356–57 Oasis HIB for, 357 SPE for, 357 sample extraction, 352–56 SBSE, 356 solvent-based, 352–54 SPE, 354–56 SPME, 355–56 supramolecular solvent-based, 353–54 sample preparation, 352 separation, 357–62 BKH Consultants, 79 BP. See Benzophenone BPA. See Bisphenol A Breast milk EDCs in, 8, 9t OT compounds analysis in, 282 PBB accumulation in, 341–42 PFASs in, 369, 372t Brominated flame retardants (BFRs), 325, 377–78. See also Flame retardants analytical methodologies, 382–93 cleanup and, 387–88 fractionation and, 387–88 instrumental, 388–93 sample preparation and, 382–87 degradation, 328 emission, 378–81 in environment, 378–81 environmental pollution, 331 in food chain, 395–402, 396t, 400t biomagnification through, 402–3 fractionation, 337 health risks/effects, 403–4 human risks, 405 physical-chemical properties, 378, 379t transformation, 378–81 Butylated hydroxyanisole (BHA), 447, 457–62, 457f analysis, 461–62 determination, 460 Butyltin compounds imposex and, 277 in seafood, 277 in urine, 282

473

C18. See Octadecylsilyl-derivatized silica Cadmium (Cd), 289 in food, 302 CALUX, 39–40 DR, 63, 68–69 Cancer breast EDCs and, 3 testicular EDCs and, 3 Capillary electrophoresis (CE), 134, 180 Captafol, 132 Carbendasim, 132 Carbon dioxide (CO2), 28–29 Carcinogenesis, OP pesticides and, 199–200 Cavitation, 26 CCA. See Cyanocobalamin CCD. See Charge-coupled device Cd. See Cadmium CE. See Capillary electrophoresis Certified reference materials (CRMs), 69 flame retardant, 394–95 hormones in food analysis and, 253 PBB determination, 343 Charge-coupled device (CCD), 294–95 Charge-injection device (CID), 294–95 Charge transfer device (CTD), 294–95 Cheese, 432 Chemicals. See also specific chemicals endocrine system and interference of, 6 hormone system and, 6 human exposure to, 8f industrial, 3 PCDDs and interference of, 61–62 PCDFs and interference of, 61–62 Chemiluminescence-based assays, OP pesticide analysis with, 211, 213, 214f Cheng disease, 12 Chlordane, 79 isomers, in animal products, 123 Chlorine herbicides, 79 Chloroacetanilide herbicides, 182 Chloroanisoles, 182 Chlorophenols, 182 Chlorpyrifos, 199, 213 Chlozolinate, 132 Chromatographic columns, 32, 205 Chromatography fungicide analysis, 134, 180 herbicide analysis, 134, 180

474

Index

Chromatography (continued) MS and, 205–6 OP pesticides in food determined with, 205–11 Chromium, 300 CID. See Charge-injection device; Collisioninduced dissociation CO2. See Carbon dioxide Codex Alimentarius Commission, 130 Cold vapor atomic fluorescence spectrometry (CV-AFS), 298, 303 Cold vapor atomic spectroscopy (CV-AS), 297–98 Collision-induced dissociation (CID), 95–96 organochlorine ED pesticides in vegetables/ fruits/cereals and, 114 Column chromatography, alkylphenolic compounds in foodstuffs purification with, 316 Combustion flames, 292 Congener separation, GC, 64–66 CONTAM. See Contaminants in the Food Chain Contaminants in the Food Chain (CONTAM), 367 Corn products, 222, 222f CRMs. See Certified reference materials CTD. See Charge transfer device CV-AFS. See Cold vapor atomic fluorescence spectrometry CV-AS. See Cold vapor atomic spectroscopy Cyanocobalamin (CCA), 330 Cyclohexane, 21, 31, 248 Cyclohexane/ethyl acetate, 248–49 Cyrodinil, 132 Daidzein, 223 Dairy products, 41 DB-5MS, 64 DBP. See Di-n-butyl phthalate DBT. See Dibutyltin DCM. See Dichloromethane DDE. See Dichlorodiphenyldichloroethylene DDT. See Dichlorodiphenyl trichloroethane Deca-BB, 328 regulatory status, 342 Deca-BBs, 327 DEHA. See Di-2-ethylhexyl adipate DEHP. See Di-(2-ethylhexyl) phthalate DEHT. See Di(2-ethylhexyl)terephthalate DES, 12

DESI-MS. See Desorption electrospray ionization mass spectrometry DESIR technique. See Dry-extraction system for IR technique Desorption electrospray ionization mass spectrometry (DESI-MS), 214 DGSANCO. See Directorate General for Health and Consumer Affairs Di-2-ethylhexyl adipate(DEHA), 258 Di-(2-ethylhexyl) phthalate (DEHP), 257 Diatomaceous earth, 24 Diazinon, 199 Dibutyltin (DBT), 278 Dichlorodiphenyldichloroethylene (DDE), 123–24. See also P,p′-dichlorodiphenyldichloroethylene Dichlorodiphenyl trichloroethane (DDT), 77–79 in products of animal origin, occurrence of, 123–24 Dichloromethane (DCM), 21, 31 Dichloromethane, in hexane, 248 2,4-Dichlorophenoxyacetic acid, 183 DiDP. See Di-iso-decyl phthalate Dieldrin, 79 DIFFERENCE project, 59 Differential pulse stripping voltammetry (DPSV), 182 Diffusion flames, 292 Di-iso-decyl phthalate (DiDP), 267 Di-iso-nonyl adipate (DiNA), 267 Di-iso-nonyl phthalate (DiNP), 267 Dimethoate, 199 ELISA for screening of, 212 DiNA. See Di-iso-nonyl adipate Di-n-butyl phthalate (DBP), 257. See also Phthalates DiNP. See Di-iso-nonyl phthalate Dioxin-like PCBs (DL-PCBs), 19 AhR binding of, 41 aquatic environment distribution of, 40 in food CALUX for, 39 threshold limits for, in EU, 42, 42t screening methods for detecting, 32 toxicity, 41 U.S. EPA regulation of, 33 Dioxins, in food, 53 Directorate General for Health and Consumer Affairs (DGSANCO), 205 Direct sample introduction (DSI), 120

Index

Disinfectants/antiseptics, 414t analysis, 424 fragment ions, 426t sample preparation, 413–19, 418t Dispersive liquid-liquid microextraction (DLLME), 132 PCP, 448 Dispersive solid-phase extraction (DSPE) MSPD and, 132–33 QuEChERS, 133 Di(2-ethylhexyl)terephthalate (DEHT), 267 Dithiocarbamate fungicides, 183 Divinylbenzene/N-vinylpyrolidone copolymer (Oasis HIB), 91 BPA sample cleanup with, 357 DLLME. See Dispersive liquid-liquid microextraction DL-PCBs. See Dioxin-like PCBs DPSV. See Differential pulse stripping voltammetry Drazoxolon, 132 DR CALUX, 63, 68–69 Dry-extraction system for IR (DESIR) technique, 183 DSI. See Direct sample introduction DSPE. See Dispersive solid-phase extraction Dynamic separation methods, 134, 180 E1. See Estrone E2. See 17β-Estradiol EC. See European Commission ECD. See Electron capture detector ECNI. See Electron capture negative ionization ED. See Electrochemical detection EDCs. See Endocrine-disrupting chemicals EDLs. See Electrodeless discharge lamps ED pesticides. See Endocrine-disrupter pesticides EDs. See Endocrine disrupters EE2. See 17α-Ethynylestradiol EFSA. See European Food and Safety Authority EI. See Electronic impact EIA. See Enzyme immunoassay Eicosanoids, 5 EI ionization. See Electron impact ionization Electrochemical detection (ED), 360 Electrodeless discharge lamps (EDLs), 297 Electron capture detector (ECD) fungicide analysis with, 180 herbicide analysis with, 180

475

PCBs in food and, 35–36 temperature and, 36 Electron capture negative ionization (ECNI), 36 Electronic impact (EI), 66 Electronic ionization, 36 Electron impact (EI) ionization, 264, 274, 274f Electrospray ionization (ESI), 95 organochlorine ED pesticides in vegetables/ fruits/cereals and, 114 Electrospray ionization-MS (ESI-MS), 223, 225f Electrothermal atomizer (ETA), 292 ELISA. See Enzyme-linked immunosorbent assay END. See Enterodiol Endocrine-disrupter pesticides (ED pesticides), 78 in food commodities, analysis of, 86–98 cleanup methods for, 92–93 fat content and, 87–88 GC for, 94 GC-MS for, 94–95 instrumental, 93–94 instrumental quantitation, 93 LC-MS, 95–96 liquid samples for, 87 matrix effects/quantitation and, 96–98 sample preparation and, 86, 87f sample pretreatment and, 86–88 solid samples for, 86–90 from liquid food matrices, extraction of, 90–92 organochlorine, 80t in food commodities, occurrence of, 121–24, 121f in products of animal origin, 114–20, 116t in vegetables/fruits/cereals, analysis of, 98–114, 98t from solid food matrices, extraction of, 88–90 ASE for, 89 MAE for, 89 MSPD for, 90 SFE for, 89–90 solvent extraction for, 88 sonication for, 89 SOX for, 88–89 Endocrine disrupters (EDs), 7–8 carcinogenic, 199–200 categorization, 79–86 chemistry, 8 corn products as, 222, 222f definitions, 7

476

Index

Endocrine disrupters (EDs) (continued) exposure concern, 86 group I, 79 group II, 86 group III, 86 risk of, 8 Endocrine-disrupting chemicals (EDCs), 3, 5–8 adverse effects of, 78, 130 in animal tissues, 130 assessing, 4 birds and effects of, 11 in breast milk, 8, 9t classification, 7 effects associated with, 10–13 endocrine disruption, 6–7 endocrine disruptors and, 7–8 endocrine system and, 5–6 in environment, 8 exposure to adverse effects of, 130 dose and, 77 human, 8–10, 8f, 9t, 10t routes of, 77–78 in fish/seafood, 78 effects of, 11 in food, 10, 246 health risks, 77 humans and effects of, 12–13 mammals and effects of, 12 marine vertebrates and effects of, 10–11 naturally occurring, 219 reptiles and effects of, 11 SDWA and screening process for, 3 SOX for, 314 suspected/known, 4t testing, 4 wildlife and effects of, 3, 5t, 10 Endocrine disruption, 6–7 alkphenolic compound, 306–7 effect, 130 fetus and, 7, 12 OP pesticide, 199, 201t Endocrine system chemical interference with, 6 EDCs and, 5–6 estrogens disrupting, 245 glands, 5 hormones, 5–6, 6f toxicity, isoflavones and, 221, 221t Endosulfan, 79, 123 Endrin, 79

ENL. See Enterolactone Enterodiol (END), 222 Enterolactone (ENL), 222 Environment antiandrogens in, 7 antiestrogens in, 7 BFRs in, 331, 378–81 EDCs in, 8 estrogens in, 7 heavy metals in, 289 PBBs in, 326–27, 340 phthalates in, 257 Environmental degradation BFR, 328 PBB, 327, 328–29 Environmental exposure, to PCP, 447 Enzyme immunoassay (EIA), 38 Enzyme-linked immunosorbent assay (ELISA), 38–39 application, 211–12 dipstick, 212 fungicide analysis and, 181–82 herbicide analysis and, 181–82 hormones in food and, 251–52 OP pesticide analysis with, 211–12 zearalenone, 238 EPA. See U.S. Environmental Protection Agency EROD, 39–40 ERs. See Estrogen receptors ESI. See Electrospray ionization ESI-MS. See Electrospray ionization-MS 17β-Estradiol (E2), 245 Estrogen(s) endocrine-disrupting, 245 environmental, 7 physicochemical properties, 245, 246t Estrogenic activity, 51 Estrogen receptors (ERs) forms, 221 isoflavones and, 219–21 phytoestrogens and, 221 Estrone (E1), 245 ETA. See Electrothermal atomizer Ethanol, 246 Ethyl acetate, 110, 246 17α-Ethynylestradiol (EE2), 245 EU. See European Union European Commission (EC), 79 DGSANCO, 205 European Food and Safety Authority (EFSA), 325

Index

European Standard Method, 64 European Union (EU) dioxin determination in food and, 53 DL-PCBs in foodstuffs, threshold limits for, 42, 42t PCBs in food and, 32, 51–52 zearalenone regulation, 232, 232t FAAS method. See Flame atomic absorption spectrometric method Famoxadone, 132 Fenitrothion, ELISA for screening of, 212 Fenthion, ELISA for screening of, 212 Ferbam. See Ferric dimethyldithiocarbamate Ferric dimethyldithiocarbamate (ferbam), 182–83 Fetus, endocrine disruption and, 7, 12 FIA. See Flow injection analysis FireMaster, 330 Fish. See also Seafood EDCs effects on, 11 EDCs in, 78 Flame atomic absorption spectrometric (FAAS) method, 182–83 Flame photometric detector (FPD), 273 Flame retardants (FRs), 377–405 analytical methodologies, 382–93, 384t cleanup and, 387–88 fractionation and, 387–88 sample preparation and, 382–87 CRMs, 394–95 in food chain, 395–402, 396t, 400t biomagnification through, 402–3 health risks/effects, 403–4 QA, 393–95 QC, 393–95 toxicity, 381–82 Florisil, 25 MSPD, 111 SPE with, 93 Florisil columns, 31 Flow injection analysis (FIA), 213 Fluorescence detection, BPA, 360 Folpet, 132 Food chain, FRs in, 395–402, 396t, 400t Food packaging, 462–64 FPD. See Flame photometric detector Fractionation, 30 FR, 387–88 PCDD, 62 PCDF, 62 Fragment ions, personal care products, 426t

477

Fungicides, 129–83 active ingredients, 130 analysis detection techniques for, 152t, 180, 180f ECD and, 180 electrochemical detection and, 182 ELISA and, 181–82 figures of merit for, 152t food matrix and, 131 GC and, 180 IAs for, 181–82 LC-MS for, 181 LC-MS/MS for, 180, 181f MS for, 181 new trends in, 131–83 analysis, sample preparation and, 131–80, 152t capillary electrophoresis for, 134, 180 CE for, 134, 180 chromatography for, 134, 180 dynamic separation methods, 134, 180 GC for, 134 HPTLC for, 134 LLE and, 131–32 MSPD, 132–33 PLE for, 180 QuEChERS, 133 SLE and, 131–32 SPE and, 131–32 TLC for, 134 USE for, 180 benzimidazolic, 132 classification, 129 definition, 129–30 dithiocarbamate, 183 in food, analysis of, 131 MRLs, 130 Gas chromatography (GC), 30. See also Multidimensional GC; 2DGC acetylation and, 250–51 congener separation, 64–66 detectors, 66–68, 67t, 273 MS, 273 ED pesticides in food commodities, analysis of, 94 FPD, 273 fungicide analysis, 134 herbicide analysis, 134 HRMS, 53 one-dimensional PCBs in food determined by, 33–34

478

Index

Gas chromatography (GC) (continued) OP pesticides in food determined with, 205–11 OT compounds analysis, 273 PAH preseparation with, 434–35, 436t HPLC v., 438–42 paraben, 455–57, 458t PBDE, 388–92 PCBs in food determined by, 33 silation and, 250 TOFMS, 95 Gas chromatography-high-resolution mass spectrometry (GC-HRMS), 280 Gas chromatography-mass spectrometry (GCMS). See also Mass spectrometry alkylphenolic compounds in foodstuffs determined with, 318 detectors for, 318 BPA, 361–62 detectors, 318 ED pesticides in food commodities, analysis of, 94–95 hormones in food, 250–51 LC-MS v., 206, 207t LODs for, 251 MS/MS v, 94–95 OP pesticide analysis with, 206 organochlorine ED pesticides in vegetables/ fruits/cereals and, 112 phthalate, 264–66, 265t, 265f phytoestrogen, 223 RTL, 113 SIM, 94 zearalenone, 234–38, 236t Gas chromatography with atomic emission detector (GC-AED), 272 GC. See Gas chromatography GC-AED. See Gas chromatography with atomic emission detector GCB. See Graphitized carbon black GC-HRMS. See Gas chromatography-highresolution mass spectrometry GC-MS. See Gas chromatography-mass spectrometry GCxGC. See 2DGC Gel permeation chromatography (GPC), 30–31, 93 alkylphenolic compounds in foodstuffs purification with, 316 column, 31 hormones in food, 247, 249 organochlorine ED pesticides, 112, 119

Genistein, 221, 221t, 223 GFAAS. See Graphite furnace atomic absorption spectrometry Glycitein, 223 Gonads, 5 GPC. See Gel permeation chromatography Graphite furnace atomic absorption spectrometry (GFAAS), 278, 293 Graphitized carbon, 24 Graphitized carbon black (GCB), 93 Haulm destructors, classification, 129 HBCD. See Hexabromocyclododecade HCB. See Hexachlorobenzene HCH. See Hexachlorocyclohexane Headspace SPME (HS-SPME), 92. See also Solid-phase microextraction phthalates, 262 Heated graphite atomizer (HGA), 292 Heavy metals, 7 dietary intake, daily, 301 environmental, 289 in food, cooking processes and, 302 in food, determination of AS, 289–303 AAS, 290–93 AES, 293–96 AFS, 296–97 AS, analytical and, 298–99 AS, sample preparation for, 299 CV-AS, 297–98 HG-AS, 298 ICP-MS, 297 selected applications of, 299–303, 300t HELIA. See Homogeneous enzyme immunoassay Hepta-BB, 328 Heptachlor, 79 Herbicides, 129–83 analysis detection techniques for, 135t, 180, 180f ECD and detection techniques for, 180 electrochemical detection and, 182 ELISA and, 181–82 FAAS and, 182–83 figures of merit for, 135t food matrix and, 131 GC and detection techniques for, 180 IAs for, 181–82 LC-MS for, 181 LC-MS/MS for, 180, 181f

Index

MS for, 181 new trends in, 131–83 other detectors for, 182–83 analysis, sample preparation and, 131–80, 135t capillary electrophoresis for, 134, 180 CE for, 134, 180 chromatography for, 134, 180 dynamic separation methods, 134, 180 GC for, 134 HPTLC for, 134 LLE and, 131–32 MSPD, 132–33 PLE for, 180 QuEChERS, 133 SLE and, 131–32 SPE and, 131–32 TLC for, 134 USE for, 180 chlorine, 79 chloroacetanilide, 182 classification, 129 definition, 130 in food, analysis of, 131 MRLs, 130 phenoxy, 132 phenoxy acid, 133 phenylurea, 132 residue analysis, 130 Hexa-BBs, 326 regulatory status, 342 toxicological studies, 330–31 Hexabromocyclododecade (HBCD), 377, 381 analytical procedures, 384t in food chain, 395–402, 400t biomagnification through, 403 health risks/effects, 404 instrumental analysis, 389t, 392–93 in seafood, 401 toxicity, 381–82 Hexachlorobenzene (HCB), 79, 122 Hexachlorocyclohexane (HCH), 79, 122–23 Hexane, 246, 261 dichloromethane, 248 Hexane/acetone, 21 MAE, 27–28 HFSLME. See Hollow fiber supported liquid membrane extraction Hg. See Mercury HGA. See Heated graphite atomizer HG-AFS. See Hydride generation-atomic fluorescence spectrometry

479

HG-AS. See Hydride generation-atomic spectroscopy High performance liquid chromatography (HPLC) alkylphenolic compounds in foodstuffs purification with, 317 zearalenone, 233–34, 235t High performance liquid chromatography mass spectrometry (HPLC-MS), 223 High-performance thin-layer chromatography (HPTLC), 134 High-pressure liquid chromatography (HPLC) PAH preseparation with, 434–35, 439t GC v., 438–41 paraben, 455–57, 458t High-resolution gas chromatography (HRGC), 32–33 High-resolution mass spectrometry (HRMS), 32–33. See also Gas chromatographyhigh-resolution mass spectrometry GC, 53 High-resolution monochromator, 294 Hollow fiber supported liquid membrane extraction (HFSLME), 132 Homogeneous enzyme immunoassay (HELIA), 252 Hormones in food analysis of, 245–53 ASE, 247 CRM for, 253 ELISA for, 251–52 GC for, 250–51 GC/MS for, 250–51 GPC, 247 HELIA for, 252 instrumental, 249–51 LC and, 250 LC/MS for, 250 MAE, 247 PSE, 247 QC for, 252–53 RIA for, 251–52 sample extraction and, 246–48 SE and, 247 systematic errors in, 252–53 TR-FLA for, 251 UAE, 247 cleanup procedures, 248–49 GPC and, 249 RAMs and, 249 SPE and, 248 human dietary exposure to, 246

480

Index

Hormone system, 5–6, 6f chemicals and, 6 functioning of, 6, 6f HPLC. See High performance liquid chromatography; High-pressure liquid chromatography HPLC-MS. See High performance liquid chromatography mass spectrometry HPTLC. See High-performance thin-layer chromatography HRGC. See High-resolution gas chromatography HRMS. See High-resolution mass spectrometry HS-SPME. See Headspace SPME Human body fluids, OT compounds analysis in, 281–82. See also Breast milk Hydride generation-atomic fluorescence spectrometry (HG-AFS), 302 Hydride generation-atomic spectroscopy (HGAS), 290 heavy metals in food determined by, 298 Hydrofurandiols, 222, 222f Hymexzole, 132 IAC. See Immunoaffinity chromatography IAs. See Immunoassays ICP. See Inductively coupled plasma ICP-MS. See Inductively coupled plasma-mass spectrometry IDMS. See Isotope dilution mass spectrometry IEH. See Institute for the Environment and Health Immunoaffinity chromatography (IAC), 62 Immunoassays (IAs), 38–39. See also Enzyme immunoassay class-specific, 212 development, 211 dipstick, 211–12 fungicide analysis with, 181–82 herbicide analysis with, 181–82 Immunochemical methods, BPA analysis, 362 Immunodyne, 212 Imposex, 269 butyltin compounds and, 277 Inductively coupled plasma (ICP), 274–76 AES, 295–96, 295f temperatures, 295–96 tubes, 295, 295f Inductively coupled plasma-mass spectrometry (ICP-MS), 290 heavy metals in food determined by, 297

Industrial chemicals, EDCs in, 3. See also Chemicals Infrared (IR) spectroscopy, 183 Insecticides classification, 129 OPC, 199 Institute for the Environment and Health (IEH), 79 Instrumental analysis, 32–33 In utero exposure endocrine disruption and, 7 to PBB, 331 In vitro damage, malathion, 199 Ion-pair reversed-phase liquid chromatography (IP-RPLC), 134 Ion-trap (IT), 95 Ion trap detectors (ITDs), 66 Ion trap mass spectrometry (ITMS), 37 Ion trap tandem mass spectrometry (ITD-MS/ MS), 37 IP-RPLC. See Ion-pair reversed-phase liquid chromatography IR spectroscopy. See Infrared spectroscopy Isofenphos-methyl, 209, 210f Isoflavones, 219–21, 220f antiestrogenic effects, 219–21 endocrine toxicity, 221, 221t ERs and, 219–21 estrogenic effects, 219 metabolism, 222–23 quantification of, in food samples, 219, 220f structure, 219, 220f in vivo effects, 219 Isooctane, 27 Isotope dilution mass spectrometry (IDMS), 56 IT. See Ion-trap ITD-MS/MS. See Ion trap tandem mass spectrometry ITDs. See Ion trap detectors ITMS. See Ion trap mass spectrometry JECFA. See Joint Expert Committee on Food Additives Joint Expert Committee on Food Additives (JECFA), 231 LC. See Liquid chromatography LC-MS. See Liquid chromatography-mass spectrometry Lead (Pb), 289 in food, 302

Index

Leukotoxindiols, 222, 222f Lignans, dietary, 221–22 metabolism, 222–23 Limits of detection (LODs), 66 GC-MS, 251 Lipid removal, 61–62 Liquid chromatography (LC) alkylphenolic compounds in foodstuffs determined with, 319 BPA, 360 MS/MS with, herbicide/fungicide analysis with, 180, 181f OT compounds analysis, 273–76 TOFMS and, OP pesticide analysis with, 206–9, 210f Liquid chromatography-mass spectrometry (LC-MS). See also Mass spectrometry alkylphenolic compounds in foodstuffs determined with, 319–20 ED pesticides in food commodities, analysis of, 95–96 fungicide analysis with, 181 GC-MS v., 206, 207t herbicide analysis with, 181 hormones in food, 250 matrix effects, 97 OP pesticide analysis with, 206 organochlorine ED pesticides in vegetables/ fruits/cereals and, 113 phytoestrogen, 223 zearalenone, 234–38, 236t Liquid-liquid extraction (LLE) ED pesticides from liquid food matrices and, 90 fungicide analysis, 131–32 herbicide analysis, 131–32 organochlorine ED pesticides, 119 OT compound analysis, 270 PCBs in food analysis and, 21–23 SOX extraction v., 23 SPE as alternative to, 91 Liquid-phase microextraction (LPME), 205, 272–73 Liquid smoke flavor (LSF), 433 LLE. See Liquid-liquid extraction LODs. See Limits of detection Low-resolution mass spectrometry (LRMS), 37 LPME. See Liquid-phase microextraction LRMS. See Low-resolution mass spectrometry LSF. See Liquid smoke flavor

481

MAE. See Microwave-assisted extraction MAES. See Microwave-assisted extraction with saponification Magnesium silicate, 24 Malathion, 199 ELISA for screening of, 212 in vitro, 199 Mammals, EDCs effects on, 12. See also Animal tissues Marine invertebrates, EDCs effects on, 10–11 Marine products, PCBs in, 40–41. See also Seafood MASE. See Microwave-assisted solvent extraction Mass spectrometric detection, 361 Mass spectrometric detection system, 112 Mass spectrometry (MS). See also specific methods chromatography and, 205–6 fragmentation pathways, 266, 266f fungicide analysis with, 181 GC detectors, 273 herbicide analysis with, 181 OT compounds analysis, 273–76, 274f PCBs in food and, 36–37 phytoestrogen analysis with, 223, 225f MAT. See Matairesinol Matairesinol (MAT), 222 Matrix effects, 96–98 Matrix-induced chromatographic response enhancement, 97 Matrix-matched standard calibration, 97 Matrix solid-phase dispersion (MSPD), 24–25 alkylphenolic compounds in foodstuffs sample extraction with, 315 DSPE and, 132–33 ED pesticides from solid food matrices and, 90 Florisil, 111 fungicide analysis, 132–33 herbicide analysis, 132–33 OP pesticide analysis with, 204–5 organochlorine ED pesticides in products of animal origin, analysis of, 118 in vegetables/fruits/cereals and, 111 PBB analysis and, 336–37 PCDDs in food and, 58 PCDFs in food and, 58 sample size, 25 steps, 25 ultrasound-assisted, 133

482

Index

Matrix solid-phase dispersion extraction (MSPDE), 309 alkylphenolic compounds in foodstuffs, analysis of, 315 Maximum contaminant level goal (MCLG), 447 Maximum residue levels (MRLs), 78, 130 MBT. See Monobutyltin MCLG. See Maximum contaminant level goal MDGC. See Multidimensional GC Meat products, PCBs in, 41 Mediterranean diet, PCBs in, 40 Mercury (Hg), 289 CV-AS, 297–98 in food, 302–3 in milk, 303 Methanol, 246, 261 Methidathion, ELISA for screening of, 212 Methylene chloride, 246 Methyl parathion, ELISA for screening of, 212 Microwave-assisted extraction (MAE), 27–28 cost, 28 ED pesticides from solid food matrices and, 89 extractants for, 27–28 hormones in food, 247 OT compound analysis, 271–72 PBB analysis and, 336 Microwave-assisted extraction with saponification (MAES), 336 Microwave-assisted solvent extraction (MASE), 60 Microwave-induced plasma atomic emission detector (MIP-AED), 274 Milk, Hg in, 303. See also Breast milk MIP-AED. See Microwave-induced plasma atomic emission detector MIPs. See Molecularly imprinted polymers Molecularly imprinted polymers (MIPs), 91 SPE and, 132 SPE sorbent, 355 SPME and, 132 Molluscicides, classification, 129 Monobutyltin (MBT), 278 Moss killers, classification, 129 MPhT. See Phenyltin MRLs. See Maximum residue levels MRMs. See Multiresidue methods MS. See Mass spectrometry MS/MS. See Tandem MS MSPD. See Matrix solid-phase dispersion

MSPDE. See Matrix solid-phase dispersion extraction MSWV. See Multiple square wave voltammetry Multidimensional GC (MDGC) columns, 34 comprehensive, 35 GC x GC, 66 heart-cut, 34–35, 65 PCBs in food and, 34–35 Multiple square wave voltammetry (MSWV), 182 Multiresidue methods (MRMs), 93 matrix-matched standard calibration, 97 organochlorine ED pesticides in vegetables/ fruits/cereals and, 110 QuEChERS method, 110–11 Musk fragrances, 414t analysis, 424–25, 425f fragment ions, 426t sample preparation, 419–20, 420t Mycoestrogens, 229–38 carcinogenicity, 231 genotoxicity, 231 metabolism, 230 regulation, 231–32 reproductive disorders in animals and, 231 toxicity, 230–31 toxicological features, 230–31 zearalenone analysis, 232–38 NDL-PCBs. See Non-dioxin-like PCBs Nerve gases, 199 Non-dioxin-like PCBs (NDL-PCBs), 19. See also Dioxin-like PCBs in food, maximum levels for, 42 Nonylphenol (NP), 306 in food, 307–8 in foodstuffs, analysis of, 318 in plastic containers, 308 VTG and, 307 Nonylphenol ethoxylates (NPEOs), 306 4-Nonylphenols, 10 NP. See Nonylphenol NPEOs. See Nonylphenol ethoxylates Oasis HIB. See Divinylbenzene/Nvinylpyrolidone copolymer Occupational Safety and Health Administration (OSHA), 447 OCPs. See Organochlorinated pesticides Octa-BB, regulatory status, 342

Index

Octadecylsilyl-derivatized silica (C18), 24–25 Octylphenol (OP), 314 Octylphenol ethoxylates (OPEOs), 306 OECD. See Organization of Economic and Cooperative Development OP. See Octylphenol OPCs. See Organophosphates OPEOs. See Octylphenol ethoxylates OPH. See Organophosphorus hydrolase OP pesticides. See Organophosphorus pesticides Organization of Economic and Cooperative Development (OECD), 4, 7 Organochlorinated pesticides (OCPs), 61 accumulation, 78 in food commodities, analysis of, 77–124 residues, 78 Organochlorine ED pesticides, 80t in food commodities, occurrence of, 121–24, 121f in fruits and vegetables, occurrence of, 122 in products of animal origin, analysis of, 114–20, 116t analytical problems, 119 fat isolation and, 115 GCxGC for, 119–20 GPC for, 119 instrumental, 119–20 LLE for, 119 MSPD for, 118 quantitation, 119–20 sample preparation and, 115–20 SFE for, 115–18 SPME for, 118 in products of animal origin, occurrence of, 122–24 in vegetables/fruits/cereals, analysis of, 98–114, 98t CID and, 114 ESI and, 114 fat content and, 111–12 GC-MS and, 112 GPC for, 112 instrumental, 112–14 instrumental quantitation, 112–14 LC-MS and, 113 MRMs and, 110 MSPD and, 111 QLIT and, 113 sample preparation for, 99–112, 100t SPE for, 112 Organochlorines, persistent, 7

483

Organophosphates (OPCs), 199–214 Organophosphorus hydrolase (OPH), 213 Organophosphorus (OP) pesticides, 199, 200f carcinogenesis and, 199–200 determination, 200 ED properties of, 199, 201t in food, analysis of, 200–201 MSPD for, 204–5 QuEChERS method for, 201–4, 204f sample treatment techniques for, 201–5, 202t SBSE for, 204–5 SDME for, 205 SPE for, 204 SPME for, 204 in food, determination of, 205–14 ambient MS for, 211, 213–14 biosensors for, 211, 212–13 chemiluminescence-based assays for, 211, 213, 214f chromatographic methods for, 205–11 ELISA for, 211–12 GC for, 205 GC-MS for, 206, 207t LC-MS for, 206, 207t LC-TOFMS and, 206–9, 210f MS/MS for, 206 nonchromatographic (fast) methods for, 211 Organotin (OT) compounds, 278 Organotin (OT) compounds analysis, 269–84 detection methods for, 273–76 GC, 273 LC, 273–76 limits of, 274, 275t MS, 273–76, 274f in human body fluids, 281–82 in other matrix, 281–83 in plastics, 283 sample preparation for, 270–76 ASE and, 271 LLE, 270 LPME, 272–73 purge and trap extraction and, 270 SPE and, 270–71 SPME, 272, 272f in seafood, 276–78, 276t samples of, 276–77, 276t seasonal variation in, 277 in seawater, 278–81 concentrations of, 280t GC-HRMS and, 280 SPME and, 278–79, 279f

484

Index

Organotin (OT) compounds analysis (continued) in sediment, 278–81 GC-HRMS and, 280 matrix effects and, 281 in sewage sludge, 281 in soils, 282–83 in textiles, 283 in various matrices, 276–83 in wastewater, 281 OSHA. See Occupational Safety and Health Administration OT compounds. See Organotin compounds Our Stolen Future (Colborn), 3 Oxadixyl, 132 PAHs. See Polycyclic aromatic hydrocarbons Pancreas, 5 Parabens, 447, 453–57 analysis methods, 455–57 chemical structure, 454, 454f detection methods, 455–57 GC methods of, 455–57, 458t HPLC methods of, 455–57, 458t in personal care products, 454 sample preparation methods, 454–55 use, 453–54 Parathion, 199 Parathyroid gland, 5 Pb. See Lead PBBs. See Polybrominated biphenyls PBDEs. See Polybrominated diphenyl ethers PCBs. See Polychlorinated biphenyls PCDDs. See Polychlorinated dibenzo-p-dioxins PCDFs. See Polychlorinated dibenzofurans PCP. See Pentachlorophenol PDAs. See Photodiode arrays PDMS. See Poly(dimethylsiloxane); Polydimethylsiloxane PDMS-DVB-coated. See Poly(dimethylsiloxane)Carbowax-divinylbenzene-coated Pentachlorophenol (PCP), 447–51, 448f analysis methods, 448–51 DLLME, 448 environmental exposure, 447 legislation, 450 in plastics, 450 SPME, 448–49 Perfluorinated compounds (PFCs), 367

Perfluoroalkylated substances (PFASs), 367–74 abbreviations, 367 cleanup, 368, 369f definitions, 367, 368t determination, 368–72, 370t in human serum/milk, 369, 372t extraction, 368, 369f in food, 372–74, 373t health aspects, 367 Perfluorooctane sulfonic acid (PFOS), 367, 368f Perfluorooctanoic acid (PFOA), 367, 368f Persistent organic pollutants (POPs), 121, 246 Persistent Organic Pollutants Review Committee (POPRC), 325 Personal care products, 413, 414t analysis, 424–27 categories, 413, 414t disinfectants/antiseptics, 413–19, 418t, 424 fragment ions, 426t musk fragrances, 419–20, 420t, 424–25, 425f parabens in, 454 preservatives/antioxidants, 420–22, 421t, 425–27 sample preparation, 413–24 sunscreen agents, 422–24, 423t Pesticides, 129–83. See also Endocrine-disrupter pesticides; Organochlorinated pesticides bioactive, 7 classification, 78 definition, 129 EDCs in, 3 OP, 199, 200f OPCs, 199–214 restricted use of, 78 PFASs. See Perfluoroalkylated substances PFCs. See Perfluorinated compounds PFE. See Pressurized fluid extraction PFOA. See Perfluorooctanoic acid PFOS. See Perfluorooctane sulfonic acid PFPD. See Pulse flame photometric detector Pharmaceuticals, EDCs in, 3 Phenoxy acid, 133 Phenthoate, ELISA for screening of, 212 Phenyltin (MPhT), 278 in urine, 282 Phosmat, ELISA for screening of, 212 Photodebromination, PBB, 329–30 Photodiode arrays (PDAs), 294 Phthalates, 257–67 analysis, contamination issue in, 261 in beverages, 262

Index

cleanup methods, 259t, 261–63 cleanup of extracts, 259t, 263–64 in environment, 257 extraction, 261–63 in fruit, 262 general structure, 258f HS-SPME, 262 instrument analysis, 264–66 GC-MS and, 264–66, 265t, 265f in milk, 262–63 MS fragmentation pathways, 266, 266f in nonfatty food, 262 physical properties, 258t sample preparation, 259t sample pretreatment, 261–63 in solid food, 263 SPME, 261–62 in vegetable oils, 262 in water, 261–62 in wine, 262 Phthalic diesters, 257 Phytoestrogens, 7, 219–25 analysis, 223, 224t, 225f corn products, 222, 222f Ers and, 221 isoflavones, 219–21, 220f lignans, dietary, 221–22 metabolism, 222–23 occurrence of, 219 Pineal gland, 5 Pituitary gland, 5 Plant growth regulators, classification, 129 Plant protection products, classification, 129 Plastics NPs in, 308 OT compounds analysis in, 283 PBBs in, 340 PCP in, 450 PLE. See Pressurized liquid extraction Poly(dimethylsiloxane) (PDMS), 204 SPME, 272 Polybrominated biphenyls (PBBs), 325–43 analytical methods, 331–40 ASE, 336 determination techniques, 337–40 liquid matrices and, 334 MAE, 336 MAES, 336 MSPD, 336–37 PLE, 336 QA and, 339

485

quantification procedures, 339–40 sample preparation techniques and, 331, 332t SFE, 335 solid matrices and, 334–37 SPME, 335–36 breast milk accumulation of, 341–42 degradation, 327, 328–29 effects of, 331 emission to air, 340 in environment, 326–27, 340 epidemiology studies, specific, 340–42 exposure to, 326 in food, analysis of, 343 historical perspective, 325–26 human exposure to, 342 long-range transport, 341 mechanisms, 328–29 occurrence, 340–42 photodebromination, 329–30 physicochemical properties, 337 in plastics, 340 production, 325–26 properties, 327–30 public concern, 325 regulatory status, 342–43 in soil, 340 solid, losses of, 340 structure, 327–30, 327f studies, 341–42 TDIs, 334 toxicological effects, 330–31 transformations, 328–29 anaerobic, 330 photolytic, 329–30, 329f in utero exposure to, 331 in wastewaters, 340 Polybrominated diphenyl ethers (PBDEs), 331, 377, 381 analytical procedures, 384t sample preparation and, 383–86 in food chain, 395–402, 396t biomagnification through, 402–3 GC for, 388–92 health risks/effects, 403–4 instrumental analysis, 388–92, 389t in seafood, 401 toxicity, 381 Poly(dimethylsiloxane)-Carbowaxdivinylbenzene-coated (PDMS-DVBcoated), 261

486

Index

Polychlorinated biphenyls (PCBs), 11, 246 in animal tissues, 25 atmospheric, 40 ban, 19 as by-product, 19 exposure to, 40 in food detecting, 35 ECD and, 35–36 EU and, 32, 51–52 GC analysis of, 33 GC analysis of, one-dimensional, 33–34 ITD-MS/MS and, 37 ITMS and, 37 MDGC and, 34–35 MS and, 36–37 TOFMS and, 37–38 in food, analysis of, 19–42 absorption columns and, 31–32 acid digestion and, 32 ASE for, 26–27 bioassays for, 39–40 cleanup and, 30 extraction and, 21, 22t fat extraction for, 21 fractionation and, 30 GPC for, 30–31 IAs for, 38–39 instrumental analysis, 32–33 liquid samples, 21 LLE for, 21–23 MAE for, 27–28 MSPD for, 24–25 other methods, 38 sample for, drying, 20 sample pretreatment and, 20–21 sample storage and, 20 saponification and, 32 solid samples, 21 SOX extraction for, 23 SPE for, 23–24 ultrasonic assisted extraction for, 26 in food, concentrations of, 40–42 distribution and, 40–41 global, 41 toxicity of, 41–42 fractionation, 337 humans and effects of, 12 in Italian diet, 23 mono-ortho, 19

non-ortho, 19 physicochemical properties, 19 Polychlorinated dibenzofurans (PCDFs). See also World Health Organization PCDD toxic equivalents accidental formation of, 51 as estrogenic substances, 51 in food, analysis of, 51–69 bioanalytical screening methods, 68–69 chemical interferences and, isolation of uncommon, 61–62 cleanup and, automation of, 62–63 cleanup methods for, 59–62 extraction and, automation of, 62–63 extraction techniques for, 56–60, 57t fractionation/group separation and, 62 GC detectors, 66–68, 67t instrumental determination, 63–68 lipid removal and, 61 MASE for, 60 MSPD for, 58 PLE for, 59–60 procedures for, 52–53 QA/QC and, 69 recovery studies and, 53–56 sample pretreatment and, 53–56 sample storage/treatment and, 53–56, 54t SFE for, 58–59 SOX extraction for, 58 SPE for, 58 spiking and recovery studies for, 56 in food, maximum levels of, 52, 52t human exposure to, 51 preventing, 51–52 humans and effects of, 12 toxicity, 51 Polychlorinated dibenzo-p-dioxins (PCDDs) accidental formation of, 51 as estrogenic substances, 51 in food, analysis of, 51–69 bioanalytical screening methods, 68–69 chemical interferences and, isolation of uncommon, 61–62 cleanup and, automation of, 62–63 cleanup methods for, 59–62 extraction and, automation of, 62–63 extraction techniques comparison and, 60 extraction techniques for, 56–60, 57t fractionation/group separation and, 62 GC detectors, 66–68, 67t instrumental determination, 63–68

Index

lipid removal and, 61 MASE for, 60 MSPD for, 58 PLE for, 59–60 procedures for, 52–53 QA/QC and, 69 recovery studies and, 53–56 sample pretreatment and, 53–56 sample storage/treatment and, 53–56, 54t SFE for, 58–59 SOX extraction for, 58 SPE for, 58 spiking and recovery studies for, 56 in food, maximum levels of, 52, 52t human exposure to, 51 preventing, 51–52 toxicity, 51 Polycyclic aromatic hydrocarbons (PAHs), 32, 429–42 in food, occurrence of, 441–42, 441t in foods/food additives, 430–31 in organism, behavior of, 429–30 preseparation procedures, 433–41 GC, 434–35, 436t GC v. HPLC, 438–41 HPLC, 435–38, 439t HPLC v. GC, 438–41 TLC, 434 sample preparation, 431–33 cheese sample treatment and, 432 LSF sample treatment and, 433 oils sample treatment and, 432–33 smoked meat sample treatment and, 431–32 Polydimethylsiloxane (PDMS), 92 Polyvinyl chloride (PVC), 257 POPRC. See Persistent Organic Pollutants Review Committee POPs. See Persistent organic pollutants Power-Prep system, 59–60 PCDD/PCFD, 63 P,p′-DDE. See P,p′-dichlorodiphenyldichloroethylene P,p′-dichlorodiphenyldichloroethylene (p,p′-DDE), 79 Preservatives/antioxidants, 414t analysis, 425–27 fragment ions, 426t sample preparation, 420–22, 421t Pressurized fluid extraction (PFE), 26, 271 Pressurized liquid extraction (PLE), 26, 271, 309 fungicide analysis, 180

487

herbicide analysis, 180 hormones in food, 247 PBB analysis and, 336 PCDDs/PCDFs in food and, 59–60 Programmable temperature vaporizer (PTV) injector, 120 Proteins, 5 PTV injector. See Programmable temperature vaporizer injector Pulse flame photometric detector (PFPD), 273 Purge and trap extraction, OT compound analysis, 270 PVC. See Polyvinyl chloride QA. See Quality assurance QC. See Quality control QLIT. See Quadrupole linear ion trap QpQ. See Triple-quadrupole Quadrupole linear ion trap (QLIT), 96, 113 organochlorine ED pesticides in vegetables/ fruits/cereals and, 113 Quality assurance (QA), 69 flame retardant, 393–95 PBB analysis, 339 Quality Assurance of Information for Marine Environmental Monitoring in Europe (QUASIMEME), 394 Quality control (QC), 69 flame retardant, 393–95 hormones in food analysis, 252–53 QUASIMEME. See Quality Assurance of Information for Marine Environmental Monitoring in Europe QuEChERS method. See Quick, easy, cheap, rugged, and safe method Quick, easy, cheap, rugged, and safe (QuEChERS) method DSPE and, 133 fungicide analysis, 133 herbicide analysis, 133 MRM, 110–11 OP pesticide analysis, 201–4, 204f pesticide residue analysis, 88 procedure, 133 sample extraction, 133 RAM. See Restricted access media Relative penis size index (RPSI), 277 Reproducibility relative standard deviations (RSDR), 302

488

Index

Reproductive disorders, 231 Reptiles, EDCs effects on, 11 Restricted access media (RAM), 91 hormones in food, 249 SPE sorbent, 355 Retention time locked (RTL), 113 RIA, hormones in food and, 251–52 RPSI. See Relative penis size index RSDR. See Reproducibility relative standard deviations RTL. See Retention time locked Safe Drinking Water Act (SDWA), 3 Saponification, 32 SBSE. See Stir bar sorptive extraction SDWA. See Safe Drinking Water Act Seafood APEOs in, 307 APs in, 307 butyltin compounds in, 277 EDCs in, 78 HBCDs in, 401 NPs in, 307 OT compounds analysis in, 276–78, 276t samples for, 276–77, 276t seasonal variation in, 277 PBDEs in, 401 preparation of, TBT and, 277–78 TBT in, 276–78 Seawater, OT compounds analysis in, 278–81 concentrations of, 280t GC-HRMS for, 280 SPME and, 278–79, 279f SEC. See Size-exclusion chromatography Secoisolariciresinol, 222 Sediment, OT compounds analysis in, 278–81 GC-HRMS for, 280 matrix effects, 281 Selected ion mode (SIM), 37 GC-MS, 94 Selected reaction monitoring (SRM), 95 Semipermeable membranes (SPMs), 61 SETAC. See Society of Environmental Toxicology and Chemistry Sewage sludge, OT compounds analysis in, 281 SFE. See Supercritical fluid extraction Silation, GC and, 250 Silica gel, 248 SIM. See Selected ion mode Simazine, 183

Single-drop microextraction (SDME), 205 Size-exclusion chromatography (SEC), 61, 264 SLE. See Solid-liquid extraction Smoked meat, 431–32 Smoking, PAHs and, 429, 442 Society of Environmental Toxicology and Chemistry (SETAC), 6 Soils, OT compounds analysis in, 282–83 Solid-liquid extraction (SLE) fungicide analysis, 131–32 herbicide analysis, 131–32 Solid-phase extraction (SPE), 23–24 alkylphenolic compounds in foodstuffs purification with, 316–17 alkylphenolic compounds in foodstuffs sample extraction with, 315 as alternative to LLE, 91 BPA, 354–56 sample cleanup and, 357 ED pesticides from liquid food matrices and, 91–92 Florisil for, 93 fungicide analysis, 131–32 herbicide analysis, 131–32 hormones in food and, 248 MIPs and, 132 OP pesticide analysis with, 204 OT compound analysis, 270–71 PCDDs/PCDFs in food and, 58 phytoestrogen, 223 sample for, 24 sorbents for, 24 MIP, 355 RAM, 355 steps, 24 Superclean LC-Si, cartridges, 248 Solid-phase microextraction (SPME), 92 alkylphenolic compounds in foodstuffs sample extraction with, 315–16 BPA, 355–56 ED pesticides from liquid food matrices and, 92 MIPs and, 132 OP pesticide analysis with, 204 organochlorine ED pesticides, 118 OT compound analysis, 272, 272f in seawater, 278–79, 279f PBB analysis and, 335–36 PCP, 448–49 PDMS, 272 phthalates, 261–62

Index

Solvent-based extraction, BPA, 352–54 Sonication, 89 SOX. See Soxhlet extraction Soxhlet extraction (SOX) alkylphenolic compounds in foodstuffs sample extraction with, 314 disadvantages, 56 EDC, 314 ED pesticides from solid food matrices and, 88–89 hormones in food, 247 LLE v., 23 PCBs in food analysis and, 23 PCDDs in food and, 56 PCDFs in food and, 56 SFE v., 30 solvents for, 23 Soxtec, 88–89 SPE. See Solid-phase extraction Spectrochemical techniques, 290 SPME. See Solid-phase microextraction SPMs. See Semipermeable membranes SRM. See Selected reaction monitoring Steam distillation, 314–15 Steroids, 5 Stir bar sorptive extraction (SBSE), 92 BPA, 356 ED pesticides from liquid food matrices and, 92 extraction phases, 205 OP pesticide analysis with, 204–5 Strata-X-AW cartridges, 248 Styrene dimers, 447, 462t in food packaging, 462–64 Styrene monomers, 464 Styrene trimers, 447, 462t in food packaging, 462–64 Sunscreen agents, 10, 414t analysis, 427 BP, 451 fragment ions, 426t sample preparation, 422–24, 423t Superclean LC-Si SPE cartridges, 248 Supercritical fluid extraction (SFE), 28–30 disadvantages, 30 ED pesticides from solid food matrices and, 89–90 instruments, 29 liquid matrix, 29 organochlorine ED pesticides

489

in products of animal origin, analysis of, 115–20 in vegetables/fruits/cereals and, 112 PBB analysis and, 335 PCDDs/PCDFs in food and, 58–59 sample drying, 29 solvents, 28 sorbent mode of, 29 SOX v., 30 Supramolecular solvent-based microextraction (SUSME), 132 BPA, 353–54 Supramolecular solvents, BPA extraction, 353–54 Surfactants, 305–20 alkylphenolic compounds, 306–7 in food matrices, 307–8 in foodstuffs, analysis of, 308–20, 309f, 310t amphoteric, 305 anionic, 305 APEOs, 305–6 APs, 305–6 aquatic toxicity, 305 cationic, 305 nonionic, 305 zwitterionic, 305 SUSME. See Supramolecular solvent-based microextraction Tandem MS (MS/MS), 94–95 DSI, 120 LC with, herbicide/fungicide analysis with, 180, 181f OP pesticide analysis with, 206 TBBP-A. See Tetrabromobisphenol A TBT. See Tributyltin TDIs. See Total dietary intakes TeBT. See Tetrabutyltin TEF. See Toxic equivalency factor TEQ. See Toxic equivalency Terbufos, 199 Tetrabromobisphenol A (TBBP-A), 377, 382 analytical procedures, 384t in food chain, 395–402, 400t biomagnification through, 403 health risks/effects, 404 instrumental analysis, 389t, 393 Tetrabutyltin (TeBT), 278 Tetramethylammonium hydroxide (TMAH), 271 Textiles, OT compounds analysis in, 283

490

Index

THBB, 329 Thin-layer chromatography (TLC). See also High-performance thin-layer chromatography fungicide analysis, 134 herbicide analysis, 134 PAH preseparation with, 434 zearalenone, 233, 234t Thiophanate-Me, 183 Thyroid gland, 5 Time-of-flight mass spectrometry (TOFMS), 37–38, 95 GC, 95 LC, OP pesticide analysis with, 206–9, 210f Time-resolved fluoroimmunoassay (TR-FLA), 251 TLC. See Thin-layer chromatography TMAH. See Tetramethylammonium hydroxide TOBB, 328 transformations, anaerobic, 330 TOFMS. See Time-of-flight mass spectrometry Total dietary intakes (TDIs), 334 Towards the Establishment of a Priority List of Substances for Further Evaluation of Their Role in Endocrine Disruption (EC-BKH 2000), 79 Toxicants, 7 Toxic equivalency (TEQ), 42 Toxic equivalency factor (TEF), 64 TPhT. See Triphenyltin TR-FLA. See Time-resolved fluoroimmunoassay Triazines, 132 Tri-BB, 328 Tributyltin (TBT), 269, 278 paints containing, 269 in seafood, 276 preparation and, 277–78 Triclocarban, 414–19, 414t Triclosan, 414–19, 414t Triphenyltin (TPhT), 269, 278 Triple-quadrupole (QpQ), 96 2DGC (GCxGC), 34–35 MDGC, 66 modulator, 35 organochlorine ED pesticides, 119–20 PCDD/PCDF, 65 UAE. See Ultrasonic assisted extraction Ultrasonic assisted extraction (UAE), 26 alkylphenolic compounds in foodstuffs sample extraction with, 309

cavitation and, 26 hormones in food, 247 solvents for, 26 Ultrasonic solvent extraction (USE) fungicide analysis, 180 herbicide analysis, 180 Ultraviolet (UV) light agents, 422 (See also Sunscreen agents) BPs and, 451 United Nations environment Programme, 325 Urine, OT compounds analysis in, 282 U.S. Environmental Protection Agency (U.S. EPA), 3 DL-PCBs in food regulated by, 33 Method 1643, 64 USE. See Ultrasonic solvent extraction UV light. See Ultraviolet light Vas deferens sequence index (VDSI), 277 VDSI. See Vas deferens sequence index Vinclozolin, 132 Vitellogenin (VTG), 251 NP and, 307 VTG. See Vitellogenin Wastewater OT compounds analysis in, 281 PBBs in, 340 WHO-PCDD-TEQs. See World Health Organization PCDD toxic equivalents Wildlife, EDCs effects on, 3, 5t, 10 Wine, 262 World Health Organization PCDD toxic equivalents (WHO-PCDD-TEQs), 52 Yusho disease, 12 Zearalenone, 229 analysis, 232–38 extraction, 233 methods of, 232 physicochemical properties and, 232–38 purification and, 233 quantification and, 233–38, 234t, 235t carcinogenicity, 231 contamination, 229 fixation, 230–31 genotoxicity, 231 metabolism, 230 origin of, 229

Index

production, 229 quantification, 233–38, 234t, 235t ELISA, 238 GC-MS, 234–38, 236t HPLC, 233–34, 235t LC-MS, 234–38, 236t TLC, 233, 234t regulation, 231–32 EU, 232, 232t

491

reproductive disorders in animals and, 231 structure, 232f toxicity, 230–31 in humans, 231 toxicological features, 230–31 Zinc ethylenebisdithiocarbamate (zineb), 182–83 Zineb. See Zinc ethylenebisdithiocarbamate Zirax, 183 Ziron, 183